Tough and Immunosuppressive Titanium-Infiltrated Exoskeleton

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Biological and Medical Applications of Materials and Interfaces

Tough and Immunosuppressive Titanium-Infiltrated Exoskeleton Matrices for Long-Term Endoskeleton Repair Seunghwan Choy, Dongyeop Oh, Seungwon Lee, Do Van Lam, Gihoon You, JinSoo Ahn, Seung-Woo Lee, Sang Ho Jun, Seung-Mo Lee, and Dong Soo Hwang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21569 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on February 3, 2019

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ACS Applied Materials & Interfaces

Type: Research Article

Tough and Immunosuppressive Titanium-Infiltrated Exoskeleton Matrices for Long-Term Endoskeleton Repair Seunghwan Choy,† Dongyeop X. Oh,‡ Seungwon Lee,† Do Van Lam,§ Gihoon You,† Jin-Soo Ahn,⊥ Seung-Woo Lee,*,†,▽ Sang-Ho Jun,*,º Seung-Mo Lee,*,§ and Dong Soo Hwang*,†

†

Department of Integrative Bioscience and Biotechnology, ▽ Department of Life Science, Pohang University of Science and Technology (POSTECH), 77 Chengam-ro, Nam-gu, Pohang 37673, Korea

‡

Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), University of Science and Technology (UST), Ulsan 44429, Korea

§

Department of Nanomechanics, Korea Institute of Machinery and Materials (KIMM), University of Science and Technology (UST), 156 Gajeongbuk-ro, Yuseong-gu, Daejeon 34103, Korea

⊥

Dental Research Institute and Department of Biomaterials Science, Seoul National University, Seoul 110-749, Korea

º Department of Dentistry, Anam Hospital Korea University Medical Center, Seoul 136-705, Korea

Keywords: chitin nanofibers, anti-inflammation, atomic layer deposition (ALD), mechanical stability, biodegradation, bone repair

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Abstract Although biodegradable membranes are essential for effective bone repair, severe loss of mechanical stability due to rapid biodegradation, soft tissue invasion, and excessive immune response remain intrinsically problematic. Inspired by the exoskeleton-reinforcing strategy found in nature, we have produced a Ti-infiltrated chitin nanofibrous membrane. The membrane employs vapor-phase infiltration of metals, which often occurs during metal oxide atomic layer deposition (ALD) on organic substrates. This metal infiltration manifests anomalous mechanical improvement and stable integration with chitin without cytotoxicity and immunogenicity. The membrane exhibits both impressive toughness (~13.3 MJ∙m-3) and high tensile strength (~55.6 MPa), properties that are often mutually exclusive. More importantly, the membrane demonstrates notably enhanced resistance to biodegradation, remaining intact over the course of 12 weeks. It exhibits excellent osteointegrative performance and suppresses the immune response to pathogenassociated molecular pattern molecules indicated by IL-1β, IL-6, and GM-CSF expression. We believe the excellent chemico-biological properties achieved with ALD treatment can provide insight for synergistic utilization of the polymers and ALD in medical applications.


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INTRODUCTION Bone repair, a major clinical challenge in the fields of periodontology and oral implantology, is a long and complicated process. It usually requires stable implant materials that are highly resistant to the biological microenvironment.1–3 Membranes for guided bone regeneration are necessary to promote bone regeneration for extended periods while also preventing rapid growth of soft tissue and invasion of the bone defect by pathogens. Patients prefer biodegradable membranes for bone repair, because they obviate the need for membrane removal surgery.4,5 Currently, materials like collagen, polyester, and chitosan are used widely in biodegradable membranes to maintain defect space and guide bone growth.6–9 However, the mechanical instability of biodegradable membranes based on these materials frequently leads to rapid biodegradation and severe immune responses, which eventually cause bone regeneration failure.10–12 Cross-linking via enzymatic, physical, and chemical treatments is commonly employed as a countermeasure for mechanically weak biodegradable membranes. However, cross-linking usually requires toxic chemical agents, such as polyepoxy compounds, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide, diphenylphosphoryl azide (DPPA), and glutaraldehyde.13 Moreover, the residual chemical agents can trigger serious cytotoxic effects or delay bone regeneration and angiogenesis.14,15 Therefore, a huge demand exists for new biomaterials that can be used to fabricate biodegradable membranes with excellent mechanical stability, good biocompatibility, proper biodegradability, and other favorable chemical and biological properties. Various organisms found in nature, such as marine worms and spiders, use minute quantities of metallic elements (e.g. Cu, Ca, and Zn) to retain the mechanical properties required for their survival.16,17 Recently, conventional atomic layer deposition (ALD) was used to intentionally infiltrate organic materials with various 3 ACS Paragon Plus Environment


metals, which led to dramatic improvements in the mechanical properties of the materials.18–21 Nevertheless, attempts to expand ALD to applications in medicine have so far been limited. Here, we report a Ti-impregnated chitin nanofibrous membrane (ChNM/Ti) that could potentially satisfy the criteria for an ideal biodegradable membrane. We infiltrated Ti into a chitin membrane by employing the vapor-phase infiltration phenomenon that is frequently observed in ALD on organic substrates.

EXPERIMENTAL SECTION Materials. Chitin (C0072) powder was purchased from Tokyo Chemical Industry Co., Ltd. and titanium isopropoxide (TIP, Ti[OCH(CH3)2]4) was purchased from Sigma. All chemicals were used without further purification. A hydrophilic polytetrafluoroethylene (PTFE) filter paper with pore diameter of 0.45 μm (HP045047D, HYUNDAI Micro., Ltd.) was used for membrane fabrication.

Preparation of ChNM/Ti by Vapor-Phase Infiltration. The chitin nanofibrous membranes (ChNMs) were loaded into an ALD reactor (S200, Savannah, Cambridge NanoTech Inc.) for multiple pulsed infiltration (MPI). The process was performed at a working temperature of 70 °C and pressure of ~0.1 torr using TIP and H2O as precursors. The ALD condition was set in exposure mode with 1 s pulse, 40 s exposure, and 60 s purging of TIP, followed by the same condition for H2O for each ALD cycle at a N2 flow rate of 30 sccm. The ALD process was repeated for 50, 100, 200, and 400 cycles for each sample. The growth rate of TiO2 on a reference Si substrate was measured as 0.5 Å/cycle.


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Electron Microscopy/Energy Dispersive Spectroscopy (EM/EDS). The formation of chitin nanofiber was confirmed by cryo-transmission electron microscopy (JEM-1011, JEOL, Japan) and high-resolution scanning electron microscopy (HRSEM, JSM-7401, JEOL, Japan). High-pressure freezing (HPF100, Leica) and subsequent freeze substitution (FS2) with a cocktail of 2% OsO4 dissolved in anhydrous acetone were used for ultrastructural analysis of the chitin nanofibrous hydrogel. For resin infiltration, Epone812 (a mixture of acetone and Spurr’s resin) was used and staining was carried out using 2% uranyl acetate and Reynolds’ lead citrate after sectioning using an ultramicrotome (MT-X, RMC, Tucson, USA). To confirm the distribution of infiltrated titanium in ChNM, the cross-sectional areas of ChNM and ChNM/400Ti were compared by EDS (51-XMX1129, Oxford Instruments) with a resolution of 5.9 keV at an accelerating voltage of 10 keV.

Raman Spectroscopy. Chemical and structural changes after ALD were analyzed from the Raman spectra, which were recorded with a Horiba Jobin-Yvon LabRam Aramis (Japan) spectrometer in the range of 200 to 2000 cm-1 with a resolution of 4 cm-1. The 785-nm line of a diode laser was used as the excitation source, and the scattered light signal was collected in a backscattering geometry using a 50× microscope objective lens. The excitation beam spot diameter was about 1 μm.

Uniaxial Tensile Test. To investigate the mechanical behaviors of various chitin membranes, uniaxial tensile tests were conducted using a universal testing machine (UTM, 3344, Instron, USA) with a 2 kN load cell and 1 mN resolution with ±0.5% uncertainty, controlled by a PC with automated-testing software. Each membrane was cut into dimensions of 4 mm × 20 mm, and their thicknesses were measured using a Digimatic Micrometer (293-240, Mitutoyo, Japan) with an accuracy of 1 μm. 5 ACS Paragon Plus Environment


The testing length and extension rate were 20 mm and 5 mm min-1, respectively. The samples were extended until fracture occurred. The force (mN)–strain (mm) data of each specimen were exported from the machine software and rescaled into an engineering stress (σ)–strain (ε) curve. For each measurement, at least nine identical samples were prepared and measured under the same conditions, and one typical data set was selected. All graphical work including data rescaling was performed with ORIGIN 9.0.

Immune Responses. Spleens were removed from C5BL/6 mice and ground through a mesh in RPMI-1640 medium (Welgene, 011-01) supplemented with 0.5% FCS (Hyclone) and 1X antibiotics (Gibco, 15240-062). To remove red blood cells, the spleen cells were suspended in RBC Blood Cell Lysing Buffer (Sigma, R7757) for 5 min at room temperature. To isolate the dendritic cells from spleen, splenic CD11cpositive DCs were enriched by positive selection with anti-CD11c monoclonal mouse antibody (BD pharmingen, 553800) magnetic beads and a MACS column (Mitenyi Biotec, Cat. 130-042-401). The spleen cells or isolated DCs (1 x 106 cells) were cultured in RPMI-1640 complete media. For LPS and CpG-ODN treatments, LPS (10 ng/ml; Sigma, L2880) or CpG ODN 1826 (1 μM; Invitrogen, tlrl-1826) was added to the cells cultured with ChNM and ChNM/400Ti for 4 h at 37 °C in an incubator. Total RNA was isolated and cDNA was synthesized with a QuantiTect Rev.Transcription Kit (QIAGEN) according to manufacturer’s protocol. Real-time PCR was performed on a 7300 Fast Real-Time PCR System (Applied Biosystems) using Power SYBR Green PCR master mix (Life technology) and primer sequences (Table S2, Supporting Information). The relative expression was determined by the cycle threshold (CΤ) method and was normalized to the internal control L32.


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Assessment of Radiographic and Histological Responses. The interface between bone and TiO2 ALD-treated ChNM was evaluated using a rat calvarial defect model by micro-computed tomography (μ-CT, SkyScan 1176, BrukermicroCT) and immunohistological staining. Before the experiments, all fifteen 9-wkold male Sprague-Dawley rats (SD rat, Orient Bio Inc.) were acclimated for 2 weeks in a specific-pathogen-free-2 (SPF-2) level facility. The rats were allowed to access the food (R+40RMM-10, SAFE) and water (Reverse osmotic water) freely. Each rat was assigned to three experimental groups such as negative control (sham), ChNM, and ChNM/400Ti in a group of five. The temperature and humidity of the facility room were automatically controlled as 23±3 ℃ and 55±10%, respectively. The day/night cycle was 12/12 h (7 am/7 pm) with an intensity of illumination of 150~300 Lux. A critical 8-mm-circular defect was formed at the calvaria of an 11-wk-old male SD rat. Each rat was anesthetized by an intraperitoneal injection of 25 mg kg-1 Alfaxan (alfaxalone, Sigma) and 15 mg kg-1 Rompun (xylazine, Bayer), and the scalp was carefully incised. The exposed cranial bone was punched using an 8-mm-diameter drill, and ChNM and ChNM/400Ti were applied to the defect; a sham surgery was adopted as a negative control. After implantation of the specimens (one specimen per rat), the incised periosteum and skin were sutured with 5-0 Vicryl sutures (Ethicon). The cross-sectional images of the interface between the implant site and bone were obtained at 12 wk with a scan of 18.02 μm for 210 ms per slice. The slice images were reconstructed as a three-dimensional picture using software (NRecon v.1.4.4, Bruker-microCT), and all the rats were euthanized by CO2 inhalation. The immunohistological responses at the interface around the implant were assessed to determine the compatibility between membrane and bone. Briefly, the cranial defect area was harvested carefully and immersed in 10% formaldehyde for 24 h to fix the tissue. Each specimen was decalcified, and the paraffin-embedded block was cut into 7 ACS Paragon Plus Environment


a 3-μm section and mounted on a glass slide. The samples were then stained with hematoxylin-eosin (H&E, Sigma), toluidine blue (TB, Sigma), and Masson’s trichrome (MT, Sigma). All the surgical procedures were approved by the Korea University Institutional Animal Care and Use Committee before conducting the animal experiments (IRB number: KUIACUC-2006-87).

RESULTS AND DISCUSSION ALD Treatment of Exoskeleton Matrices. We applied TiO2 ALD using titanium isopropoxide (Ti[OCH(CH3)2]4, TIP) as the first precursor and H2O as the second precursor to chitin membranes isolated from crustacean exoskeletons, as shown schematically in Figure 1b. To prepare the chitin membranes, we first ground refluxed chitin in water to obtain partially deacetylated chitin nanofibers with a ~37% degree of deacetylation. We then fabricated the chitin nanofibrous membranes (ChNM) (Figure S1 and S2).22

Characterization of Ti-Infiltrated ChNM. Cryogenic transmission electron microscopy (cryo-TEM) analysis of the dispersed nanofibers (Figure 2a) revealed they were similar to natural nanofibers with diameters of ~6.2 nm. Their diameters were increased to ~27.4 nm by the reconstitution of interfibrillar hydrogen bonds during ChNM formation. The impregnation of Ti into the bulk following vapor-phase infiltration by TiO2 ALD was confirmed by field-emission scanning electron microscopy (FE-SEM, JSM-7401, JEOL, Japan) and EDS analysis (Figure 2b). The EDS signal intensity from infiltrated Ti was high in chitin treated with 400 cycles of TiO2 ALD (ChNM/400Ti), particularly at the surface, which was directly exposed in the ALD chamber. The deposited TiO2 layer on the surface was imaged by TEM, which is shown in Figure 2c. The chemical bonds presumed to have formed between 8 ACS Paragon Plus Environment

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impregnated Ti and chitin were examined with Raman and Fourier-transform infrared (FTIR) spectroscopy. As can be seen in Figure 2d for Raman spectra, the intensities of the peaks at 1450 and 1416 cm-1, corresponding to the rocking vibration of CH2, increased following Ti infiltration. The ratio of the intensities of the two characteristic peaks at 1376 and 1369 cm-1, which were also attributed to CH2 rocking, changed due to conformational variance. The reaction of TIP with amine and hydroxyl groups was verified by the peaks at 270 cm-1 and 800 cm-1, which were indicative of ν(Ti-N)23 and ν(Ti-O-C)24 in the Raman and FTIR spectra, respectively (Figure S3). However, it should be noted that formation of the Ti-N bond is energetically unfavorable. Therefore, the Ti-N bond may have formed less readily than the Ti-O, Ti-O-N (TiOxNy), and Ti-C bonds.25,26 The intensities of the C-O-C and C-O stretching bands at ~1059 cm-1, which were attributed to oxygens at the C1, C5, and C6 positions of the glucosamine-based ring27, decreased. The peak intensities of the amide I, II, and III vibrational modes also decreased due to steric hindrance and the chemical reaction with introduced Ti. Based on the IR data, it appeared that TIP reacted with amine and hydroxyl groups at an early stage. As they are Lewis bases, these groups are excellent electron donors. Reaction with TIP thus disrupted the strong intermolecular hydrogen bonds and glucosamine-based ring backbone structures (Figure 2e).28

Anomalous Mechanical Properties and Resistance to Biodegradation. The infiltrated Ti increased the mechanical stability of the chitin membranes and prolonged the biodegradation time. Various chitin membranes treated with different numbers of TiO2 ALD cycles were prepared, and uniaxial tensile tests were performed. The representative stress-strain curves in Figure 3a show that the slopes observed at an initial strain of ~0.5% markedly decreased as strain increased with all 9 ACS Paragon Plus Environment


of the Ti-infiltrated chitin membranes. This was possibly because of changes in the molecular structures of the membranes as their amorphous contents increased. However, the Ti-infiltrated chitin membranes exhibited greater strength and toughness than the raw chitin membrane (Figure 3b). The ChNM/400Ti membrane in particular exhibited greatly enhanced tensile strength (55.64 ± 1.92 MPa) and toughness (13.3 ± 0.21 MJ∙m-3) (Figure S4). It is worth noting the toughness of the Tiinfiltrated chitin membrane was several times greater than that of a commercial collagen GBR membrane, although it was less than that of generic engineered polymers. The mechanical properties of crystalline polymeric materials are largely determined by the size and orientation of their crystallites and their amorphous content.29,30 Crystal structure analysis performed by X-ray diffraction (XRD) indicated the crystalline structure of chitin became highly distorted upon Ti infiltration (Figure S5 and Table S1). The characteristic peaks of chitin appeared at 9.4º, 19.6º, 22.8º, 25.8º, and 39º, which reflected the (020), (110), (130), (013), and (063) planes.22 Provocative titanium infiltration resulted in the disappearance of the (130), (013), and (063) peaks, suggesting serious distortion of the chitin crystal structure. Based on these observations, it appeared that a small amount of Ti gave rise to marked changes in the microstructure of chitin, which resulted in a drastic increase in the mechanical stability of the chitin membrane. Interestingly, Ti infiltration significantly enhanced biodegradation resistance. In-vivo evaluation of biodegradation in ChNM/400Ti with the rat calvarial defect indicated that degradation time was notably prolonged. In the millimeter-scale histological data, the raw ChNM exhibited horizontal degradation through enzymatic and hydrolytic processes (Figure 3c). However, the ChNM/400Ti membrane remained almost intact when exposed to the lytic activity of the physiological environment (Figure 3d). This suggested the Ti


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infiltration process could be usefully applied as an efficient, non-toxic cross-linking method with various polymers for practical clinical applications.

Evaluation of Immunogenicity and Immune Suppression. Biologically, the TiO2 layer on the surface of ChNM/400Ti elicited an attractive immune response and displayed good osteointegration performance. Tissue metabolism is extensively affected by inflammatory cytokines, particularly following implantation of foreign materials.31,32 To examine whether the chitin nanofibrous materials had inherent immunogenicity or immune-modulating activity, we co-cultured both ChNM and ChNM/400Ti with total spleen cells (SCs) or fluorescence-activated cell sorting (FACS)-sorted dendritic cells (DCs) with typical pathogen-associated molecular pattern (PAMP) molecules, such as lipopolysaccharide (LPS) and CpG-DNA, which are ligands of toll-like receptors (TLRs). We then measured expression of the following inflammatory cytokines and colony stimulating factors: IL-1β, IL-6, TNF-α, IFN-α, IFN-β, IFN-γ, M-CSF, GM-CSF, and G-CSF. In general, ChNMs elicited no stimulatory activity in SCs and DCs without PAMP stimulation, which indicated the absence of inherent immunogenicity in these materials (Figure 4a–c, Figure S6a–c, and Table S2). Surprisingly, ChNMs suppressed the production of inflammatory cytokines when the immune cells were stimulated by PAMPs. The immunosuppressive activity of ChNMs differed depending on the type of immune cell, i.e. unseparated SCs or purified DCs. ChNM/400Ti exerted stronger inhibition than did ChNM when the materials were exposed to SCs and DCs along with the TLR agonists. With regard to GM-CSF and G-CSF, Tiinfiltration appeared to further upregulate the immunosuppressive effects of ChNM, suggesting synergistic action between ChNM and Ti in the material (Figure 4c). In



contrast, the IL-6 and IFN-γ data indicated that inhibitory effects of ChNM only occurred when Ti had infiltrated the material (Figure 4a,b). It is worth noting that ChNM/400Ti affected the production of a broad range of cytokines in the stimulated SCs, which included the most inflammatory cytokines. These were the typical pro-inflammatory cytokines IL-1 β, IL-6, and TNF-α; type I & II IFNs; and colony stimulating factors (CSFs). These results suggested that ChNM/400Ti may have affected the upstream signaling pathways of TLRs. It is has been established that TLRs are closely connected with bone remodeling. TLR2mediated upregulation of a nuclear factor kappa-Β ligand (RANKL) receptor activator in Staphylococcus aureus has been reported to stimulate bone resorption and osteoclastogenesis.33 In terms of immune response, these features seem to cause premature failure of implants, which is due primarily to vigorous inflammation. Moreover, cytokine secretion patterns are intimately linked to osteointegration over time. For example, IL-6 and TNF-α present in the activated inflammatory state can induce osteoclastogenesis with local bone resorption, and IL-1β inhibits bone morphogenetic protein expression and collagen synthesis.34,35 Additionally, IFN-α, β, and γ activate osteoclastogenesis through specific signaling cascades, which are intimately connected to TLRs.36 Lee et al. suggest that GM-CSF is crucial for the fusion of mononuclear osteoclasts through the promotion of Ras/ERK pathway activation, thereby directing bone resorption.37 Nevertheless, additional studies are needed to determine how TiO2–ALD treatment of ChNM affects innate immune responses in detail. Although the immune-stimulating effects of TiO2 have been reported38–40, our results suggest that ALD treatment may modify the immunological properties of ChNM to bring about a novel immunosuppressive effect. The immunosuppressive properties of ChNM/400Ti might be further elucidated by studying the combined effects of chitin and Ti derivatives. 12 ACS Paragon Plus Environment

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Performance in Bone Repair and Osteointegration. Bioactivity and biocompatibility of the Ti-infiltrated membranes were confirmed by the in-vitro proliferative behaviors of MC3T3-E1 preosteoblasts and NIH3T3 fibroblasts over 7 d (Figure 5a). Amorphous TiO2 deposited on the ChNM/400Ti surface elicited more proliferative activity of the preosteoblasts than did commercially available machined and sandblasted/acid-etched (SLA) titanium surfaces. Active preosteoblastic proliferation is the first prerequisite for the formation of bone-like nodules and initially requires a large volume of cells.41 After cell proliferation, differentiation is required if the cells are to be incorporated into a tissue, which includes bone maturation. Moreover, ChNM/400Ti coated with a TiO2 layer enabled active growth of fibroblasts, thus demonstrating that Ti ALD-treated chitin was not cytotoxic and enhanced bioactivity. These properties could be helpful for dermal healing at a surgical site. In our radiographic and histological evaluations, the negative control groups (sham surgery) exhibited a limited bone growth length ratio (~0.47) at the critical defect site, which was due to invasive soft tissue in the absence of osteoconductive substances (Figure 5b,c). We observed little new bone growth, and soft tissue filled the entire defect site. This was because the soft tissue had a faster growth rate than that of bone tissue. In the case of ChNM, guided bone growth was insufficient but better than that of the negative control due to the inherent bioactivity of chitin. The resulting bone contained osteoblasts (Ob), osteocytes (Os), lamellar bone (LB), and mature woven bone (WB), which confirmed the metabolic activity of bone tissue. In addition, we observed a bone growth length ratio of ~0.52 (Figure 5b,d). However, only new bone adhering to the original tissue contained woven and lamellar bone. Woven and lamellar bone were not observed at locations distant from the original bone. This 13 ACS Paragon Plus Environment


indicated the remodeling process for bone repair was not effective without adjacent new bone. The unsatisfactory bone guiding ability of the exoskeleton was improved by the surface TiO2 layer on ChNM/400Ti. Osteoid (Oi), Ob, Os, LB, and WB were observed in the newly formed bone, which was well-developed across the whole defect site and bridged the distance between the two original bones. In addition, slanted bone growth occurred owing to its high affinity for ChNM/400Ti, which is known as osteointegration. Slanted bone growth was indicated at the interface by the enhanced bone growth length ratio (~0.72), which was greater than that observed in the sham and ChNM samples by 53% and 42%, respectively (Figure 5b,e). This implied that the TiO2 layer on ChNM/400Ti afforded well-guided bone growth by increasing affinity at the bone-chitin interfaces. During bone repair, the sequential processes of blood clotting, replacement with granulation tissue, and osteoblastic osteoid formation must occur before biomineralization can begin.42 We confirmed the emergence of osteoids in newly formed bone only in the ChNM/400Ti group, in which active bone remodeling was still taking place at 12 weeks. The biomineralization of osteoids is manifested by randomly oriented collagen fibers (woven bone) and lamellar bone formed with woven bone templates. We therefore assumed that biomineralization of osteoids occurred with assistance from the chitin and Ti derivatives and resulted in bone repair. Nevertheless, it will be necessary to conduct a future study in larger animals, such as rabbits, to monitor biological responses to ChNM/400Ti over periods greater than 12 weeks.

CONCLUSIONS In summary, by employing the vapor-phase infiltration process that occurs frequently during metal oxide ALD on organic substrates, we successfully produced 14 ACS Paragon Plus Environment

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Ti-impregnated chitin nanofibrous membranes. The Ti-impregnated membrane had excellent toughness (~13.3 MJ∙m-3) and high tensile strength (~55.6 MPa), which are often mutually exclusive properties. More importantly, the membrane demonstrated markedly enhanced resistance to biodegradation and remained intact over the 12week study. It also displayed excellent osteointegration and immunosuppressive effects. We believe the impressive chemico-biological properties of the Tiimpregnated membrane may produce better clinical outcomes for bone repair than other biodegradable membranes currently used in clinical applications. Exploiting the ALD technique for substantive medical applications will also provide insight into how it can be employed to synergistically improve mechanical and biological functionality.

Supporting Information Additional experimental and characterization details can be found in the Supporting Information. Figures S1-S2 show the fabrication process for chitin nanofibers and the degree of deacetylation determined from the conductometric titration curve. Figures S3-S6 provide physicochemical data (FT-IR, statistical evaluation of tensile tests, XRD) and immunological information for cytokines from sorted dendritic cells. Table S1 summarizes d-spacing and nanocrystallite size. Table S2 contains the details of qPCR primer sequences.

Corresponding Authors *E-mail: [email protected] (S.W.L) *E-mail: [email protected] (S.H.J) *E-mail: [email protected] (S.M.L) *E-mail: [email protected] (D.S.H)



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Author contributions: D.S.H., D.X.O., and S.M.L. conceived the idea. S.C. and D.V.L. performed sample preparation and characterization. S.L., G.Y., J.S.A., S.C., and S.H.J. performed the immunological and in-vivo experiments. S.C., D.X.O., D.S.H., S.W.L., and S.M.L. wrote and edited the manuscript.

ACKNOWLEDGMENTS We would like to acknowledge the financial support from a Marine Biotechnology program grant (Marine BioMaterials Research Center) funded by the Ministry of Oceans and Fisheries of Korea (D11013214H480000110), the National Research Foundation

of

Korea

Grant

funded

by

the

Korean

Government

(NRF-

2016M1A5A1027594 & NRF-2017R1A2B3006354), and the Basic Science Research Program through the National Research Foundation, funded by the Ministry of Education (NRF-2017R1D1A1B03028418).

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(20) Lee, S. M.; Pippel, E.; Moutanabbir, O.; Gunkel, I.; Thurn-Albrecht, T.; Knez, M. Improved Mechanical Stability of Dried Collagen Membrane after Metal Infiltration. ACS Appl. Mater. Interfaces 2010, 2, 2436-2441. (21) Gong, B.; Peng, Q.; Jur, J. S.; Devine, C. K.; Lee, K.; Parsons, G. N. Sequential Vapor Infiltration of Metal Oxides into Sacrificial Polyester Fibers: Shape Replication and Controlled Porosity of Microporous/Mesoporous Oxide Monoliths. Chem. Mater. 2011, 23, 3476-3485. (22) Ifuku, S.; Nogi, M.; Abe, K.; Yoshioka, M.; Morimoto, M.; Saimoto, H.; Yano, H. Preparation of Chitin Nanofibers with a Uniform Width as α-Chitin from Crab Shells. Biomacromolecules 2009, 10, 1584-1588. (23) Du, H.; Xie, Y.; Xia, C.; Wang, W.; Tian, F. Electrochemical Capacitance of Polypyrrole–Titanium Nitride and Polypyrrole–Titania Nanotube Hybrids. New J. Chem. 2014, 38, 1284-1293. (24) Musschoot, J.; Xie, Q.; Deduytsche, D.; Van den Berghe, S.; Van Meirhaeghe, R. L.; C. Detavernier. Atomic Layer Deposition of Titanium Nitride from TDMAT Precursor. Microelectron. Eng. 2009, 86, 72-77. (25) Caubet, P.; Blomberg, T.; Benaboud, R.; Wyon, C.; Blanquet, E.; Gonchond, J.; Juhel, M.; Bouvet, P.; Gros-Jean, M.; Michailos, J.; Richard, C.; Iteprat, B. LowTemperature Low-Resistivity PEALD TiN Using TDMAT under Hydrogen Reducing Ambient. J. Electrochem. Soc. 2008, 155, H625-H632. (26) Zhang, H.; Lv, X.; Li, Y.; Wang, Y.; Li, J. P25-Graphene Composite as a High Performance Photocatalyst. ACS Nano 2010, 4, 380-386. (27) Ehrlich, H.; Maldonado, M.; Spindler, K.; Eckert, C.; Hanke, T.; Born, R.; Goebel, C.; Simon, P.; Heinemann, S.; Worch, H. First Evidence of Chitin as a Component of the Skeletal Fibers of Marine Sponges. Part I. Verongidae (Demospongia: Porifera). J. Exp. Zool. B 2007, 308, 347-356. 19 ACS Paragon Plus Environment


(28) Kumirska, J.; Czerwicka, M.; Kaczynski, Z.; Bychowska, A.; Brzozowski, K.; Thoming, J.; Stepnowski, P. Application of Spectroscopic Methods for Structural Analysis of Chitin and Chitosan. Mar. Drugs 2010, 8, 1567-1636. (29) Galeski, A.; Strength and Toughness of Crystalline Polymer Systems. Prog. Polym. Sci. 2003, 28, 1643-1699. (30) Al-Sawalmih, A.; Li, C.; Siegel, S.; Fabritius, H.; Yi, S.; Raabe, D.; Fratzl, P.; Paris, O. Microstructure and Chitin/Calcite Orientation Relationship in the Mineralized Exoskeleton of the American Lobster. Adv. Funct. Mater. 2008, 18, 3307-3314. (31) Gristina, A. G. Biomaterial-Centered Infection: Microbial Adhesion Versus Tissue Integration. Science 1987, 237, 1588-1595. (32) Raphel, J.; Holodniy, M.; Goodman, S. B.; Heilshorn, S. C. Multifunctional Coatings to Simultaneously Promote Osseointegration and Prevent Infection of Orthopaedic Implants. Biomaterials 2016, 84, 301-314. (33) Kassem, A.; Lindholm, C.; Lerner, U. H. Toll-like Receptor 2 Stimulation of Osteoblasts Mediates Staphylococcus Aureus Induced Bone Resorption and Osteoclastogenesis through Enhanced RANKL. PLoS One 2016, 11, e0156708. (34) Arron, J. R.; Choi, Y. Osteoimmunology-Bone Versus Immune system. Nature 2000, 408, 535-536. (35) Ma, Q. L.; Zhao, L. Z.; Liu, R. R.; Jin, B. Q.; Song, W.; Wang, Y.; Zhang, Y. S.; Chen, L. H.; Zhang, Y. M. Improved Implant Osseointegration of a Nanostructured Titanium Surface via Mediation of Macrophage Polarization. Biomaterials 2014, 35, 9853-9867. (36) Takaoka, A.; Taniguchi, T. New Aspects of IFN-α/β Signalling in Immunity, Oncogenesis and Bone metabolism. Cancer Sci. 2003, 94, 405-411.


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Figures and Captions

Figure 1. Chitin membrane and Ti infiltration. (a) Schematic illustration of the crab exoskeleton, a natural chitin/inorganic hybrid structure embedded as a nanofibrous template in a protein matrix. (b) ChNM/Ti hybrid prepared by the vapor-phase infiltration phenomenon (see text for details).


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Figure 2. Chemical changes in chitin after Ti infiltration. (a) Cryo-TEM image of fabricated chitin nanofibers dispersed in water (~6.2 nm diam.) and FE-SEM image of ChNM prepared by vacuum filtering and drying (~27.4 nm diam.). (b) Cross-sectional FE-SEM micrograph and EDS map of ChNM and ChNM/400Ti (arrow: dense Ti signal, inset: histogram result). (c) TEM image of TiO2 layer (~18 nm thick) deposited on the ChNM surface at 150,000X magnification. (d) Raman spectra of chitin before and after Ti infiltration. Green boxes indicate noteworthy changes. (e) Presumable chemical structure of chitin after Ti infiltration.



Figure 3. Changes in mechanical stability and relevant biodegradation properties before and after Ti-infiltration. (a) Stress-strain curves of the raw chitin membrane and chitin membranes treated with different numbers of TiO2 ALD cycles. (b) Ashby plot (toughness vs. tensile strength) comparing the mechanical stability of our Tiinfiltrated chitin membrane to that of commercial collagen GBR membranes and engineered polymers. In-vivo evaluation of biodegradation of (c) ChNM and (d) ChNM/400Ti after 12 weeks. The cyan arrows indicate failure of horizontal integration.


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Figure 4. Real-time PCR data showing levels of cytokine expression in total spleen cells. (a) Pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α); (b) interferons (IFN-α, β, and γ); and (c) colony-stimulating factors (M-CSF, GM-CSF, and G-CSF). Data are shown as the mean ± SD. All experiments were performed in triplicate (n = 3), and significant differences were confirmed statistically (*p < 0.05, **p < 0.01, ***p < 0.001, and undetected, UD).



Figure 5. Biological activity and biocompatibility of ChNM/Ti. (a) In-vitro proliferation (7 d) of MC3T3-E1 preosteoblasts and NIH3T3 fibroblast cells on two commercial titanium samples, ChNM, and ChNM/400Ti. (b) Radiographic analysis of guided bone growth calculated in the sagittal plane by the ratio of bone length [(L+R)/F]. Histological assessments of new bone growth and osteointegration for (c) negative control (sham surgery), (d) ChNM, and (e) ChNM/400Ti. Each tissue specimen was observed after staining: (i) toluidine blue (1.5X magnification), (ii) Masson’s trichrome (400X magnification), and (iii) hematoxylin and eosin staining (400X magnification). M, membrane; ST, soft tissue; NB, new bone; OB, original bone; LB, lamella bone; Ob, osteoblast; Os, osteocyte; WB, woven bone; DM, dura mater; OI, osteointegration. All


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experiments were performed in triplicate (n=3; mean value ± SD), and significant differences were confirmed statistically (*p < 0.05 and **p < 0.01).



Graphical Abstract


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Graphical Abstract 145x68mm (150 x 150 DPI)

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Figure 1. Chitin membrane and Ti infiltration. (a) Schematic illustration of the crab exoskeleton, a natural chitin/inorganic hybrid structure embedded as a nanofibrous template in a protein matrix. (b) ChNM/Ti hybrid prepared by the vapor-phase infiltration phenomenon (see text for details). 265x236mm (150 x 150 DPI)


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Figure2. Chemical changes in chitin after Ti infiltration. (a) Cryo-TEM image of fabricated chitin nanofibers dispersed in water (~6.2 nm diam.) and FE-SEM image of ChNM prepared by vacuum filtering and drying (~27.4 nm diam.). (b) Cross-sectional FE-SEM micrograph and EDS map of ChNM and ChNM/400Ti (arrow: dense Ti signal, inset: histogram result). (c) TEM image of TiO2 layer (~18 nm thick) deposited on the ChNM surface at 150,000X magnification. (d) Raman spectra of chitin before and after Ti infiltration. Green boxes indicate noteworthy changes. (e) Presumable chemical structure of chitin after Ti infiltration. 247x230mm (150 x 150 DPI)



Figure 3. Changes in mechanical stability and relevant biodegradation properties before and after Tiinfiltration. (a) Stress-strain curves of the raw chitin membrane and chitin membranes treated with different numbers of TiO2 ALD cycles. (b) Ashby plot (toughness vs. tensile strength) comparing the mechanical stability of our Ti-infiltrated chitin membrane to that of commercial collagen GBR membranes and engineered polymers. In-vivo evaluation of biodegradation of (c) ChNM and (d) ChNM/400Ti after 12 weeks. The cyan arrows indicate failure of horizontal integration. 369x204mm (150 x 150 DPI)


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Figure 4. Real-time PCR data showing levels of cytokine expression in total spleen cells. (a) Proinflammatory cytokines (IL-1β, IL-6, and TNF-α); (b) interferons (IFN-α, β, and γ); and (c) colonystimulating factors (M-CSF, GM-CSF, and G-CSF). Data are shown as the mean ± SD. All experiments were performed in triplicate (n = 3), and significant differences were confirmed statistically (*p < 0.05, **p < 0.01, ***p < 0.001, and undetected, UD). 260x256mm (150 x 150 DPI)



Figure 5. Biological activity and biocompatibility of ChNM/Ti. (a) In-vitro proliferation (7 d) of MC3T3-E1 preosteoblasts and NIH3T3 fibroblast cells on two commercial titanium samples, ChNM, and ChNM/400Ti. (b) Radiographic analysis of guided bone growth calculated in the sagittal plane by the ratio of bone length [(L+R)/F]. Histological assessments of new bone growth and osteointegration for (c) negative control (sham surgery), (d) ChNM, and (e) ChNM/400Ti. Each tissue specimen was observed after staining: (i) toluidine blue (1.5X magnification), (ii) Masson’s trichrome (400X magnification), and (iii) hematoxylin and eosin staining (400X magnification). M, membrane; ST, soft tissue; NB, new bone; OB, original bone; LB, lamella bone; Ob, osteoblast; Os, osteocyte; WB, woven bone; DM, dura mater; OI, osteointegration. All experiments were performed in triplicate (n=3; mean value ± SD), and significant differences were confirmed statistically (*p < 0.05 and **p < 0.01). 496x459mm (150 x 150 DPI)


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Tough and Immunosuppressive Titanium-Infiltrated Exoskeleton

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