Accepted Manuscript “The return of ceramic implants”: Rose stem inspired dual layered modification of ceramic scaffolds with improved mechanical and anti-infective properties Chen Li, Fanrong Ai, Xinxin Miao, Hang Liao, Fengshun Li, Mingzhuo Liu, Fen Yu, Lina Dong, Ting Li, Xiaolei Wang PII: DOI: Reference: S0928-4931(17)34684-2 doi:10.1016/j.msec.2018.08.044 MSC 8838 To appear in: Materials Science & Engineering C Received date: Revised date: Accepted date: 1 December 2017 6 July 2018 20 August 2018 Please cite this article as: Chen Li, Fanrong Ai, Xinxin Miao, Hang Liao, Fengshun Li, Mingzhuo Liu, Fen Yu, Lina Dong, Ting Li, Xiaolei Wang , “The return of ceramic implants”: Rose stem inspired dual layered modification of ceramic scaffolds with improved mechanical and anti-infective properties. Msc (2018), doi:10.1016/ j.msec.2018.08.044 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT “The return of ceramic implants”: rose stem inspired dual layered modification of ceramic scaffolds with improved mechanical and anti-infective properties PT Chen Lia,#, Fanrong Aib,c,e,#, Xinxin Miaoa, Hang Liaoa, Fengshun Lib, a SC RI Mingzhuo Liud, Fen Yu b, Lina Dong b, Ting Lib & Xiaolei Wanga,b* Department of Orthopedic Surgery, The Second Affiliated Hospital of b NU Nanchang University, Nanchang, Jiangxi, 330006, China. Institute of 330031, China. c MA Translational Medicine, Nanchang University, Nanchang, Jiangxi, School of Mechanical & Electronic Engineering, D Nanchang University, Nanchang, Jiangxi, 330031, China. d Department of PT E Burns, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, 330006, China. eKey Laboratory of Lightweight and high CE strength structural materials of Jiangxi Province, Nanchang University, AC Nanchang 330031, China. #These authors contributed equally to this work.* Address correspondence to E-mail: email@example.com Abstract Nowadays, traditional ceramics for bone implants have considerably replaced by metal based biomedical materials, attributing to the friability of ceramics. However, ceramic implants possess excellent biocompatibility and longtime abrasion resistance. They should be more ACCEPTED MANUSCRIPT desirable for long-term uses of implants in case their fragility had been overcome. In the present work, inspired from natural rose, a dual-layer-modified ceramic scaffold was constructed by coating a superplastic layer of isocyanate (ISO) resin and a nano Zinc Oxide PT (nano-ZnO) layer on the ceramic scaffold. The ISO resin modification RI layer with 1 mm thickness, improved the mechanical properties of SC ceramic implants 2-3 times, and protect the ceramic implants from broken even drop from 1 meter high. Moreover, such dual layered modification NU exhibited broad spectrum antibacterial behavior. In vivo biocompatible MA studies demonstrated that there was no obvious noticeable tissue damage D in all major organs of mice after the implant surgeries. PT E Keywords: Surface modification; Ceramic implants; 3D printing; Antibacterial activity; Biomedical engineering CE 1. Introduction AC Great progresses of biomaterials for bone implants has been made in the past decades [1-4]. According to the bioactivities of biomaterials, artificial bone implants can be mainly divided into three types: biotolerant materials (titanium, tantalum, ceramic, etc), bioactive materials (bioglass, hydroxylapatite, etc.) and reabsorbable biomaterials (polylactic acid, polyglycolic polymers and processed bone grafts etc) . However, biotolerant materials are still the most widely used in clinic at present, ACCEPTED MANUSCRIPT attributing to their excellent mechanical properties and biocompatibility. However, An ideal artificial bone implants for long-term use should possess not only favorable mechanical properties and biocompatibility, but high resistance to corrosion and friction [6, 7]. Metallic biomaterials PT such as titanium are the most used artificial bone implants in clinic [8, 9]. RI However, debris generating from frequent friction are almost inevitable SC after long-term usage (around 10 years) of metallic implants. The accumulation of these debris can easily lead to aseptic inflammation and NU implant-associated osteolysis, resulting in prosthesis-loosening which can MA only be solved through artificial joint revision surgery [10-12]. Theoretically, pure ceramic biomaterials possess a significant high D abrasion resistance. They should be more desirable for long-term uses of PT E various implants, especially for joint replacement implants. This have confirmed by many literatures and clinical results in recent years [13-15]. CE However, ceramic biomaterials have their intrinsic defective property of AC fragility, hampered their clinical application [16, 17]. Besides, since most of ceramic biomaterials are difficult to be implanted through minimally invasive surgery, the risk of post-operative infection also cannot be ignored . Significantly improved mechanical properties and broad spectrum antibacterial capacity are still the challenges for ceramic implant as an ideal implants. Bioinspired from the structure of nature rose, a dual layered ACCEPTED MANUSCRIPT modified ceramic implant was constructed to overcome above drawbacks of fully ceramic implants in present study. A superplastic layer of ISO resin was modified on the surface of ceramic implants to enhance its mechanical properties, and a nano-zinc oxide (ZnO) slices as the second PT layer were surface modified to prevent the implants from bacterial RI invasion. Microstructure and mechanical properties of the dual layered SC modified ceramic implant were performed. Moreover, in vitro and in vivo Biocompatibility and antibacterial properties were investigated. NU 2. Experimental Section MA 2.1. Fabrication of ZnO-I&R dual layered modified ceramics. Yttrium oxide-stabilized zirconium oxide (3Y-ZrO2) nano-powder D was used to prepare two different shapes of ceramics by gel-casting PT E method. Briefly, an aqueous mixture of 3Y-ZrO2 nano-powder and ammonium citrate was prepared and pH was adjusted to 9 with ammonia. CE Then acrylamide, N,N'-methylene bisacrylamide, ammonium persulfate AC and tetramethylethylenediamine were added. The ceramic slurry was slowly poured into 3D-printed molds which were realized by 3D software CATIA and 3D printer (Makerbot Z18, America), followed with gelation and drying at temperature of 55 ℃ for two days in vacuum. The obtained green bodies were sintered at 1200 ℃ forming 3Y-ZrO2 ceramics. Then ISO resin were uniformly coated on the ceramics by a thermal spray coating process under pressure of 2000 psi and temperature of 65 ℃, with ACCEPTED MANUSCRIPT the thickness of the ISO resin layer was controlled at about 1mm. Nano-zinc oxide solution was synthesized by adding 50 mM Zn(NO3)2﹒ 6H2O, 80 mM NH3﹒H2O and 25 mM hexamine into 400mL deionized water, followed by 85 ℃ water bath for 24 h. Then the nano-zinc oxide PT solution was dropped on ISO resin coated ceramics, forming the ZnO- RI ISO dual layered modified ceramics . SC 2.2. Characterization The crystalline patterns of ZrO2 nano-powder sintered at different NU temperatures (600 ℃, 800 ℃, 1000 ℃ and 1200 ℃, respectively) were MA analyzed by X-ray diffraction (Rigaku D/max 2550 ， Japan). Then DSC-TG analysis of ZrO2 ranging from 30 ℃ to 1300 ℃ was recorded D using thermogravimetry (Netzsch STA, Germany). The morphology of PT E unmodified ceramic, ISO resin coated ceramic and ZnO- ISO dual layered modified ceramic were examined by scanning electron CE microscope (SEM) (Zeiss, Germany). And the nano-zinc oxide was AC analyzed by transmission electron microscopy (TEM) (Zeiss, Germany). Mechanical properties of samples in bending test, tensile test and compressive test were evaluated by a WDW series electronic universal testing machine (Hualong, China). Impact mechanical property was performed on a CBD series auto controlled impact testing machine (Hualong, China) to accomplish the impact test. 2.3. Antibacterial Properties Assay in vitro ACCEPTED MANUSCRIPT Antibacterial properties of ISO resin coated ceramic and ZnO- ISO dual layered modified ceramic were analyzed. Unmodified ceramics were set as control group. All samples were sterilized by ethylene oxide before test. This study was processed against two of the most common clinical PT strains: Staphylococcus aureus (S.aureus, gram positive, ATCC 25923) RI and Escherichia coli (E.Coli, gram negative, ATCC 25922). Five SC milliliters of Luria−Bertani broth, 100 μl of bacterium suspension and sample were mixed in sterile tube and cocultured in an orbital shaker for NU 24 h. After that, 100 μl of the coculture solution was taken out and diluted MA 105 times. Then 50 μl of the media was used to coat on Petri dish which was placed in a constant temperature incubator (37 °C) for 24 h. Plate PT E samples . D counting method was used to compare antibacterial potency of different 2.4. Cytotoxicity Assay CE Cytotoxicity of ISO resin was analyzed by cell counting kit-8 AC (CCK-8) assay. Briefly, sterile ISO resin coated ceramic was soaked into cell culture medium as a ratio of 1cm2 material to 1mL cell culture medium according to the standard procedures in ISO10993. The mixture was incubated in a cell culture incubator containing 5% CO2 at 37 ℃. After 24 h, the solution was collected as extract concentrate and diluted by the cell culture medium to ratios of 1/2, 1/4, 1/8, 1/16. Then rat bone marrow mesenchymal stem cells (rBMMSCs) were seeded in 96-well ACCEPTED MANUSCRIPT plate at an initial density of 5×103 cells/well and cultured in different concentrations of the extracts. The extracts were changed in every two days. The cell culture medium without extract addition was used as blank group. rBMMSCs were continuously cultured for 7 d and CCK-8 assay PT was applied to detect cell OD values at time point of 1, 3 and 7 d RI respectively according to manufacturer's instructions. Enzyme-linked SC immunosorbent assay microplate reader (Synergy 2, Bio-TEK) was used to measure OD value of each well at a wavelength of 450 nm which NU reflected the number of alive cells . MA 2.5. In Vivo Biocompatibility Studies The toxicity of ISO resin was conducted by directly contacting with D mouse tissue (24 h toxicity test) and tail vein injecting of ISO resin PT E extract solution with PBS (14 d toxicity test). All animal use procedures were according to the NIH guide for the Care and Use of Laboratory CE Animals and were approved by the local Care Committee. AC 24 h toxicity test: All surgeries were performed under aseptic conditions, with sterile surgical instruments and operating table. After 8 h fasting period, the mice were given anesthesia intraperitoneally with 3% pentobarbital sodium solution (Sigma) at a dosage of 1.0 mL/kg . A full thickness incision (length = 1.0 cm) was created from the dorsum on each mouse. Sterile ISO resin (10 mm × 10 mm × 2.5 mm) was placed subcutaneously, directly contacting with skin and muscle in experimental ACCEPTED MANUSCRIPT group while the control group was taken operation only. After 24 h, all mice were sacrificed by cervical dislocation. The tissues of surgical site were removed for further histological analysis (hematoxylin and eosin staining, H&E staining). The specimens dehydrated with a graded series PT of ethanol were embedded in paraffin and sectioned (approximately 5 m RI thickness), stained with hematoxylin and eosin, then analyzed under a SC light microscope [23, 24]. 14 d toxicity test : Sterile ISO resin was soaked into fresh NU phosphate buffered solution (PBS) as the proportion of 1 cm2/mL for 24 h. MA The extract (0.1 mL) was directly injected into experimental group mice by caudal vein injection once a day, lasting for 14 d while the control D group were injected with PBS (0.1 mL) only. The mice were carefully fed PT E for 14 d, afterwards, all mice were anesthetized with ether. Blood samples obtained by cardiac puncture were collected for biochemical test. Then CE the mice were sacrificed by cervical dislocation and immediately AC eviscerated. The hearts, livers, spleens, lungs and kidneys removed from the mice were fixed in 10 % neutral buffered formalin for further histological analysis (H-E staining). Blood samples were immediately sent to be done biochemical analyses. 3. Results and Discussion 3.1 Preparation of ZnO- ISO dual layer modified ceramic In order to improve the mechanical and antibacterial properties of ACCEPTED MANUSCRIPT ceramic implants, a ZnO- ISO dual layer modified ceramic implant was constructed bioinspired from the structure of nature rose. As depicted in Figure 1A, some relative slender parts which are similar with the inner parts of rose stem are easy to be damaged (As shown Figure 1B). While PT this brittle area of rose was protected by a dual layered coverage: rose RI cortex and rose thorn. The rose cortex can remarkably improve the SC physical strength of the stem, and the thorns can effectively protect the rose from biological attacks. Based on this inspiration, a similar dual NU layered modification was constructed for slender ceramic rod (Figure 1C): MA the most fragile area of ceramic was firstly coated with a plastic layer of ISO resin to enhance mechanical properties of ceramic implants. Then D nano-zinc oxide (ZnO) slices were surface modified, as the second layer, AC CE PT E preventing the implants from bacterial invasion. Figure 1. Schematic of a dual layered modification of ceramic scaffolds. (A) Integrative ceramic biomaterial has many advantages, but some slender parts of it are too fragile, limited its further use. (B) The idea of the proposed dual layered modified ceramic was inspired from ACCEPTED MANUSCRIPT rose. Cortex and thorns, the dual layered structure of rose, protected inner rose stem. (C) Due to the rose inspiration, dual layered modified ceramic was constructed. The first layer was ISO Resin, organic coating on ceramic. Then nano-ZnO slices as the second layer covered on the surface PT of ISO modified ceramic. RI Zirconia ceramics are biocompatible and have been shown to have SC higher fracture toughness and bending strength than other ceramics, making them suitable as materials for bone implants or dental implants. good clinical outcomes . Hence, we chose yttrium MA with NU They have extensively been used as ball heads in total hip replacements oxide-stabilized zirconium oxide (3Y-ZrO2) ceramics as the ceramic D implants for dual layered modification. 3Y-ZrO2 ceramics with different PT E shapes were fabricated by gel-casting method, and then sintered at different temperature. To ensure the stability of this ceramic biomaterial, CE DSC-TG analysis (Figure S1A) was used to study its changes at different AC heating stages (ranging from 0 ℃ to 1300 ℃). As a result, no significant variation on the TG value of ZrO2 was observed during the entire heating process. On the other hand, the volatilization of organic components and water led TG value falling down from 100 % to 97.95 % which illustrated ZrO2 involved in this experiment was stable. There was a peak within the range of 300-400 ℃, indicating crystal transformation of ZrO2 occurred. Afterward, different ZrO2 samples sintered at 600 ℃, 800 ℃, 1000 ℃ and ACCEPTED MANUSCRIPT 1200 ℃ were then analyzed through XRD respectively (Figure S1B). Results revealed the uniform and well crystal growth of ZrO2. No extra crystalline phase was found. Therefore, the sintering temperature of 1200 ℃ was selected for ZrO2 samples preparation. PT 3D printed technique was employed to construct appropriate shaped RI slot molds (Figure 2A), and then uniform columnar and rod-like ceramics SC were accordingly fabricated by gel-casting process (Figure 2B) for mechanical tests. Figure 2B and Figure 2C indicated all ceramics had NU smooth appearance. Then ISO resin layers were modified on the surfaces MA of two different shaped ceramics (Figure 2D and Figure 2E), respectively. It is worthy to note that ISO resin material possessed high quality, certain D elasticity and smooth surface without peculiar smell. The thickness of AC CE subsequent tests. PT E ISO resin material on ceramics was set as approximately 1mm for the Figure 2. Different optical images show that the fabricated samples ACCEPTED MANUSCRIPT were uniform. (A) 3D-printed long slot molds. (B) Rod-shaped ceramics. (C) Columnar ceramic. (D) Rod-shaped ceramics sprayed with ISO resin layer. The coating thickness was adjusted to approximate 1mm. (E) Columnar ceramics sprayed with ISO resin layer. PT The relative mechanical properties on 3Y-ZrO2 ceramics and ISO RI resin modified ceramics were summarized in Figure 3. Four different SC groups of the tests were carried out, including impact test (Figure 3A), bending test (Figure 3B), tensile test (Figure 3C) and compressive test NU (Figure 3D). Impact test showed that, after merely 1mm modification, MA ISO resin modified ceramics (1.47±0.11 J) could withstand two times more impact force than the unmodified ceramics (0.62±0.09 J) (Figure D 3A). In bending test, the ISO resin modified ceramics (23.72±9.01 N) had PT E three times higher bending force resistance than unmodified ceramics (7.10±2.48 N) (Figure 3B and Figure S2). Tensile test also revealed the CE ISO resin modified ceramics (18.03±0.76 Mpa) had more anti-tensile AC capacity than unmodified ceramics (11.56±1.70 Mpa) (Figure 3C and Figure S2). Another straightforward demonstration was provided in the form of an interesting video section: both unmodified ceramic and ISO resin modified ceramic were dropped from hands of a robot simultaneously. The control group shattered into different pieces of various shapes while the experimental group was undamaged (The video segment was provided in the supplementary information). Above results ACCEPTED MANUSCRIPT proved that the mechanical properties of ISO resin modified ceramics were significant enhanced. It was worth noting that in impact test, the unmodified ceramics had (Figure 3F, f) not only exhibited worse mechanical properties, but also showed sharply irregular fracture section. PT As a comparison, the fracture section of experimental group (Figure 3E, e) RI was smooth and regular. This phenomenon was also shown in bending SC test and tensile test. This phenomenon is main caused by the superplastic properties of ISO resin layer. Owning to the intrinsic fragility of ceramics, NU they tend to be broken instantly and show a sharp fracture section when MA subject to impact force. After the modification of superplastic ISO resin, the impact force can be absorbed by ISO resin layer quickly and then then D gradually released on the ceramics, resulting a smooth fracture section PT E and higher force resistance of ISO resin modified ceramics. Considering ceramic artificial joint might be fractured by some serious accidents, CE there is a potential risk to take difficult revision surgery in clinic when AC several small sized pieces of implant were remained in the surrounding tissue. ISO resin material modified ceramics with improved mechanical properties and regular fracture section could be a better alternative. Another interesting phenomenon was observed in compressive test. Although, there was no significant difference between two groups in compressive test (Figure 3D). The main reason might be ISO resin material couldn’t provide supportive force. But the ceramics in control ACCEPTED MANUSCRIPT group (Figure 3H) were instantly fractured leading numerous ceramic pieces. If this process occurred in human, the tissues surrounding ceramics would be damaged severely. On the other hand, the inner ceramics in experimental group (Figure 3G) were also completely PT compressed into pieces, but the outer ISO resin layer kept the integrity of RI the ceramic shapes and ceramic pieces were not harmful to the AC CE PT E D MA NU SC surroundings. Figure 3. Mechanical tests. (A-D) Four kinds of mechanical tests were implemented: impact test (p***<0.001), bending test (p*<0.05), tensile test (p***<0.001) and compressive test (p>0.05). (E-F) Cross sections of the ceramics after impact test. (e-f) The inserts images are the high magnification of the red circle selected area. The purple arrows in inserts image (f) point to irregular cross section in control group. (G-H) Optical ACCEPTED MANUSCRIPT images of different group ceramics after compressive test. 3.2 Antibacterial properties of dual layered modified ceramic In order to endow ceramic implants with antibacterial properties, nano-ZnO surface modification was further preformed. Figure 4A-C PT showed the SEM morphology of unmodified ceramic, I&R material RI coated ceramic and ZnO- ISO dual layered modified ceramic respectively. SC It is demonstrated that the ceramic was well coated by ISO resin and the nano-ZnO slices were evenly distributed on the surface of ISO resin NU coated ceramic. TEM image of the nano-ZnO slices (Figure 4D) showed MA the average size of as-synthesized nano-ZnO slices was about 100nm. Wafers were selected as the model substrates to quantitatively investigate D the antibacterial performance of ISO resin modified ceramics and PT E ZnO-ISO dual layered modified ceramic, and unmodified ceramic was set as control group. ISO resin shows no inhibition on the growth of both CE S.aureus (gram positive bacteria) and E.Coli (gram negative bacteria) at AC all (Figure 4E, F and S3). No obvious different was found on the numbers of bacterial colonies between ISO resin modified ceramic and unmodified ceramic. In contrast, ZnO-ISO dual layer modified ceramic exhibited excellent antibacterial activities against both S.aureus and E.Coli (Figure 4G, H and S3). Almost no bacterial colonies formed when bacteria were treated with ZnO-ISO dual layered modified ceramic. RI PT ACCEPTED MANUSCRIPT SC Figure 4. Morphology of modified ceramics and antibacterial NU properties assay in vitro. SEM images of unmodified ceramic (A), ISO resin modified ceramic (B) and ZnO-ISO dual layered modified ceramic MA (C). The images were processed by pseudo color, and each color represented different composition: yellow (ceramic), green (ISO resin), PT E D red (ZnO nanoslices). (D) TEM image of ZnO nanoslices. (E-H) Antibacterial studies of different ceramic coatings against E.coli and CE S.aureus in vitro, for ISO resin modified ceramic (p>0.05) and ZnO-ISO dual layered modified ceramic (p***<0.001). AC 3.3 Biocompatibility Studies of dual layered modified ceramic Since ceramic is known to have well biocompatibility and zinc oxide is the only nano-sized antibacterial material certified by the FDA in vivo [27, 28], the present cytotoxicity study was mainly focused on the biocompatibility of ISO resin layer. Cytotoxicity test (CCK-8) revealed the rat bone marrow mesenchymal stem cells could be suppressed only under 7 days’ continuous culture in high concentrations of extracts (50% ACCEPTED MANUSCRIPT and 100%), while other conditions no obvious influence on cell growth was found (Figure 5A). But such extreme condition would not occur in human body as normal metabolism of body fluid existed. 24 h toxicity test of ISO resin to mice was further studied in vivo (Figure 5B-D). PT Results showed that, after contacting with ISO resin for 24 h, there was RI no macroscopic abnormalities such as swelling, hemorrhage, necrosis in AC CE PT E D MA NU SC skin or muscle (Figure 5D). ACCEPTED MANUSCRIPT Figure 5. Cytotoxicity assay and 24 h toxicity test. (A) Cytotoxicity of ISO resin material was analyzed by CCK-8 assay. (B-D) The optical diagram of mice in 24 h toxicity test. (E-H) The tissues (skin and muscle) around the ISO resin implant materials were stained by H&E method in PT 24 h toxicity test. RI Furthermore, skin and muscle of mice were sliced and stained by SC H&E for pathological examination. Results in Figure 5E-H further revealed that there was no noticeable tissue abnormality. Additionally, as NU showed in Figure 6, within 14 days’ continuous observation, mice of both MA experimental group and control group showed normal daily activities and diets. There was no significant difference in H&E histologic analysis of D major organs (heart, liver, spleen, lung and kidney) (Figure 6 A-J) and PT E blood tests (TBIL, ALT, AST, ALP, UREA and CREA) (Figure 6 K-P). All the results proved that ISO resin material had excellent CE biocompatibility for contacting tissues and important organs, without AC influencing morphology and function of them. D MA NU SC RI PT ACCEPTED MANUSCRIPT PT E Figure 6. 14 d toxicity test. (A-J) The mice organs (heart, liver, spleen, lung and kidney) were stained by H&E method in 14 d toxicity test. (K-P) CE Biochemical analyses (TBIL, ALP, CREA, UREA, ALT, AST) of mice AC blood samples were shown in 14 d toxicity test 4. Conclusions Infection is still a critical problem affecting the therapeutic effects of ceramic implants. Meanwhile, fragility is a key problem for the wide use of ceramics. Aiming to overcome these two disadvantages, we inspired from natural rose and developed ZnO-ISO dual layered modification on 3Y-ZrO2 ceramics implants. Further experimental results demonstrated ACCEPTED MANUSCRIPT that, the proposed dual layered modification could not only enhance mechanical properties, but also endow antibacterial ability. Considering the great biocompatibility of this dual layered modification, we believe the current study could promote the revival of several ceramic based PT biomaterials. However, the long-term effects of ZnO-ISO dual layered RI modified ceramic implant in the body, are still unknown. More systematic SC in vivo study of ZnO-ISO dual layered modified ceramic is needed before further clinical application. NU Acknowledgments MA This work was supported by the National Natural Science Foundation of China (No.21461015 to Wang Xiaolei); the Science Foundation of D Jiangxi Provincial Department of Education (No.KJLD14010 and PT E 20153BCB23035 to Wang Xiaolei); the major program of Natural Science Foundation of Jiangxi Province (No.20161ACB21002 to Wang CE Xiaolei); the Foundation of Health and Family Planning Commission of AC Jiangxi Province (No. 20155246 to Li Chen); the Science Foundation Jiangxi Provincial Department of Traditional Chinese Medicine (No. 2013A243 to Li Chen); the Foundation of The Second Affiliated Hospital of Nanchang University (No. 2016YNZJ12007 to Li Chen); China Postdoctoral Science Foundation (No. 2017M610402 to Ai Fanrong); Postdoctoral Science Foundation of Jiangxi Province (No. 2017KY06 to ACCEPTED MANUSCRIPT Ai Fanrong); LINE-X(CHINA) Company is appreciated for the technical support for ISO resin surface modification. 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Lars S, Amir D, Birgitta L, Mattias I. Bone Tissue Responses to Surface-Modified Zirconia CE 26 Implants: A Histomorphometric and Removal Torque Study in the Rabbit. Clinical Implant Dentistry and Related Research.7 (2005) S13-S20. Wang X, Zhu H, Bao Y, Yang F, Yang X. Uniform hierarchical frameworks patterned by movable AC 27 magnetic microparticles. Acs Nano. 5 (2011) 3250-3256. 28 Wang X, Zhu H, Yang F, Yang X. Biofilm‐Engineered Nanostructures. Advanced Materials. 21 (2009) 2815-2818. ACCEPTED MANUSCRIPT Author Contributions X.L.W. conceived and designed the experiments. C.L. and F.R.A. PT performed the sample fabrication involved in this experiment, characterization, cytotoxicity assay and data analysis and wrote the RI manuscript. X.X.M. and H.L. accomplished the mechanical tests. F.S.L. SC and M.Z.L. accomplished the antibacterial properties assay in vitro. F.Y. NU and L.N.D. contributed to biocompatibility studies in vivo. T.L. revised the paper. X.L.W. is corresponding author. All authors discussed the MA results and commented on the manuscript. Additional information PT E D Supplementary information accompanies this paper. Conflict of interest: The authors declare no conflicts of interest for this AC CE article. AC CE PT E D MA NU SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Graphical Abstract Inspiration of the dual layered modification of ceramic scaffolds from AC CE PT E D MA NU SC RI PT natural rose ACCEPTED MANUSCRIPT Highlights: Inspired from natural rose, a dual-layer-modified ceramic scaffold was constructed by coating isocyanate resin and nano Zinc Oxide layers on the ceramic scaffold. The proposed dual-layer-modification significantly improved their mechanical AC CE PT E D MA NU SC RI PT properties and their broad spectrum antibacterial capacity.