Mussel-Inspired Peptide Coatings on Titanium Implant to Improve

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Tissue Engineering and Regenerative Medicine

Mussel-Inspired Peptide Coatings on Titanium Implant to Improve Osseointegration in Osteoporotic Condition Huan Zhao, Yingkang Huang, Wen Zhang, Qianping Guo, Wenguo Cui, Zhiyong Sun, David Eglin, Lei Liu, Guoqing Pan, and Qin Shi ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00261 • Publication Date (Web): 26 May 2018 Downloaded from http://pubs.acs.org on May 26, 2018

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ACS Biomaterials Science & Engineering

Mussel-Inspired Peptide Coatings on Titanium Implant to Improve Osseointegration in Osteoporotic Condition Huan Zhao†,∆.‡ Yingkang Huang, †,∆.‡ Wen Zhang, †,∆.‡ Qianping Guo, ∆ Wenguo Cui, ∆ Zhiyong Sun, † David Eglin,€ Lei Liu, § Guoqing Pan,§,* Qin Shi †,∆,£,* †

Department of Orthopaedics, The First Affiliated Hospital of Soochow University, 188 Shizi St,

Suzhou, 215006, China ∆

Orthopedic Institute, Soochow University, 708 Renmin Rd, Suzhou, 215007, China

£

Key Laboratory of Stem Cells and Biomedical Materials of Jiangsu Province and Chinese

Ministry of Science and Technology, 199 Renai Rd, Suzhou, 215123, China €

AO Research Institute Davos, Clavadelerstrasse 8, Davos, 7270, Switzerland

§

Institute for Advanced Materials, School of Materials Science and Engineering, Jiangsu

University, Zhenjiang, Jiangsu 212013, China

† These authors contributed to this work equally.

* Corresponding authors: Fax/Tel: +86-512-67781420, +86-512- 67781169 E-mail: [email protected] (G. Pan) [email protected] (Q. Shi)

KEYWORDS: Titanium implant, mussel-inspired peptide, osteoporosis, osteogenesis, osseointegration, catechol-metal coordination.

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ABSTRACT: Osteoporosis greatly impairs in vivo implant osseointegration due to the poor osteogenesis in osteoporotic condition and the low bioactivity of implants such as titanium-based biomaterials. Various surface engineering strategies, including unstable physical absorption or complex chemical conjugations, have been developed to biofunctionalize titanium implants and improve interfacial osseointegration. However, very few of them took into consideration the clinically challenging osteoporotic condition as well as dual-functionalization of the implants for improvement of both osteoblast adhesion and osteogenesis. In this work, we combined two mussel-inspired bioactive peptides (i.e., with cell adhesive or osteogenic sequences) for one-step dual-functionalization of Ti screws via a facile self-organized multivalent coordinative interaction. In vitro study indicated that the biomimetic dual-functional coating could efficiently improve the osteogenesis of osteoporosis-derived mesenchymal stem cells despite of their impaired bone metabolism. Moreover, under osteoporotic in vivo condition, the dual-functional peptide coating on Ti screws could also give rise to significant enhancement of interfacial osteogenesis, newly formed bone condition, osseointegration as well as implant mechanical stability. This is probably due to the integrin-targeted cell adhesive and osteogenic motifs on the modified Ti screws, which recovered the regular bone metabolism equilibrium between osteogenesis and osteoclastogenesis in an osteoporotic condition. We anticipate that the highly biomimetic peptides and one-step dual-functionalized strategy would provide a facile and effective means for improving the clinical outcome of Ti-based implants in patients with a disturbed bone metabolism.

1. Introduction Efficient osseointegration at the bone-implant interface, i.e., the formation of direct and stable bone-to-implant connection, plays a very important role in the clinical success of endosseous


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implants.1,2 This suggests the ideal implants should possess bioactivities to induce cell adhesion to enhance anchoring or promote osteogenicity to accelerate interfacial bone regeneration.3-6 As the most popular implant biomaterials, titanium (Ti) has been widely used in orthopedics and odontology, due to their excellent mechanical property and biochemical stability in vivo.7 However, the major problem of Ti implants is low bioactivity or bio-inert nature, and on their own, lack of direct bioactivities to induce cell anchoring or bone formation at the bone-implant interfaces.8 Nevertheless, the clinical outcome of direct use of Ti implants (i.e., without introducing cell adhesive or osteogenic factors) seems to be acceptable. In fact, this result mainly benefits from the tissue self-repair ability of organisms, in which the healthy bone metabolism actually is helpful to form a relatively stable bone layer between the implants and surrounding bone tissues. Unfortunately, such kind of bare implants are more prone to failure for the patients who are suffering from metabolic diseases such as osteoporosis.9 Osteoporosis is a representative bone metabolic disease in humans over 50-year-old, which is characterized by the imbalance between bone formation (osteogenesis) and bone resorption (osteoclastogenesis).10, 11 Due to decreased bone mineral density and deteriorated trabecular bone microstructure, bone fracture is prevalent in osteoporotic patients.9, 12 Although internal fixation based on endosseous screw implants is an effective for the treatment of bone fracture, significantly higher incidence of unstable fixation arising from screw loosening and cutout was reported in patients with osteoporosis.13-17 The crux is the poor osteogenesis in osteoporotic patients and subsequently weak osseointegration on the interface of screw and bone tissues, which can only withstand low pull-out strength. It is also worth mentioning that, some approaches based on optimizing the surface properties of Ti implants including roughness and macro-/nano-structures18-24 could improve osseointegration in non-disturbed bone metabolic


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conditions. Such positive effects, however, have been barely reported in an osteoporotic condition only by virtue of surface geometry and topology. This fact suggests the necessity of introducing desirable bioactivities (e.g., inducing osteoclast adhesion or the interfacial osteogenesis) on a Ti implant to improve osseointegration in vivo,25 especially under an osteoporotic condition. Up to now, various bioactive molecules, ions or composites have been employed for surface modification of Ti implants. For example, osteoconductive hydroxyapatite (HA) layers26-28 and the calcium phosphate (CaP) coatings29, 30 have been deposited on implant surfaces and also proven to promote early osseointegration. However, the non-uniformity and delamination risk of these inorganic biomineralized coatings as well as the complicated fabrication technologies greatly limited their clinical applications.31,

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In addition, cell adhesive factor RGD peptide

(Arg-Gly-Asp), 33-36 extracellular matrix components (e.g., collagen, fibronectin and laminin), 28, 37-41

osteoinductive growth factors (bone morphogenetic proteins, BMP),40, 42-45 osteoinductive

drugs (e.g., alendronate and zoledronate)46-48 and ions (Sr and Zn)49-51 have been covalently immobilized or physically adsorbed on the surface of implants to improve osseointegration. These surface engineering strategies, however, are suffering from either the burst molecular release in physical absorption models or the fairly complex procedures in chemical conjugations.52-55 Moreover, very few of these studies took into consideration the clinically challenging condition such as osteoporosis32, 46-48 as well as the demand of multi-biofunction56 on an idea endosseous implant, i.e., inducing both osteoclast adhesion and interfacial osteogenesis. Inspired by Mytilus edulis foot proteins, we previously designed two biomimetic catecholcontaining peptides with cell adhesive peptide RGD53, 57 or osteogenic growth peptide (OGP) YGFGG58 for one-step biofunctionalization of Ti implants.59 The mussel-inspired peptides could


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spontaneously and stably bind on Ti implants via strong coordination interactions between catechol groups on the peptide and titanium oxide on the Ti surface60 and subsequently the peptide-functionalized Ti implant exhibited significantly improved cell adhesion, osteogenicity and osseointegration both in vitro and in vivo. This is ascribed to the improved biological response including early recruitment, attachment, proliferation and differentiation of bone cells on the Ti implant surface. However, the peptides we designed then were too hydrophobic, which are not well soluble in PBS. In this study, we designed and synthesized another two musselinspired peptides with more hydrophilic sequences (SSSSS) as the spacer to improve their solubility in PBS as well as their accessibility for cell receptors recognition. We speculate that the mussel-inspired, one-step biofunctionalization strategy by using such kind of biomimetic active peptides would also exhibit great potential to improve Ti implants osseointegration under osteoporotic condition (Scheme 1). We will apply the new bioactive mussel-inspired peptides ((DOPA)4-S5-GRGDS and (DOPA)4-S5-YGFGG) for mono- or dual-functionalization of medical Ti screws and evaluate the effect of biomimetic peptide coatings on osseointegration by using the ovariectomized (OVX) rats as osteoporotic animal model.61 We will focus on the evaluation of 1) in vitro osteogenesis of the osteoporosis-derived stem cells on the biomimetic peptide coated surfaces, and 2) in vivo osseointegration of peptide coated Ti screw implants in osteoporotic animal model, for example, interfacial bone healing, histological observation and finally the mechanical stability. Insert Scheme 1 2. Experimental Sections 2.1 Materials


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Two mussel-inspired peptides with cell adhesive motif and osteogenic growth factor (DOPA)4-S5-GRGDS (RGD-peptide) and (DOPA)4-S5-YGFGG (OGP-peptide), respectively were synthesized according to previously reported method.59 Phosphate buffer saline solution (PBS, 0.02 mmol/L, pH=7.2) prepared with MilliQ water, purified with a Thermo Scientific Barnstead NANOpure Diamond Water Purification Systems to give a minimum resistivity of 18.2 MΩ·cm and a purchased phosphate buffer salt (Beyotime Biotechnology, China). TiO2coated quartz substrates with 80-100 nm TiO2 layer (10 mm or 15 mm in diameter) were provided by the Center for NanoChemistry, Peking University (Beijing, China). Medical cortical bone self-tapping screw (HAQ04, Material model T, 1.5 mm × 6 mm) were purchased from Waston Medical Appliance Co., Ltd. (Changzhou, China). Other used reagents are reported in the description of the experiment. 2.2 Isolation of bone marrow mesenchymal stem cells (MSCs) Bone marrow mononuclear cells from healthy 6-week-old Sprague–Dawley (SD) rats or osteoporotic rats by ovariectomy (See details in Section 2.6) were collected and incubated in Dulbecco’s modified Eagle’s medium-low glucose (DMEM-LG, Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). After cultured for 24 h, the adherent mesenchymal stem cells (MSCs) were left by replacing the medium. MSCs from healthy rats (H-MSCs) and from osteoporotic rats (OP-MSCs) were confirmed by flow cytometric analysis (Guava EasyCyte 6HT; Millipore, Billerica, MA, USA) of CD29, CD90, CD34 and CD45 (fluorescein isothiocyanate- or phycoerythrin-conjugated antibodies; eBioscience, San Diego, CA, USA). 2.3 Mussel-Inspired Peptide Coatings


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Before peptide coating, the TiO2 modified quartzes or cortical bone self-tapping Ti screws were first cleaned by oxygen plasma treatment for 60s. Then, the quartzes or cortical bone selftapping Ti screws were immersed in (DOPA)4-S5-YGFGG and (DOPA)4-S5-GRGDS, or their mixed PBS solutions in molar ratio at a total peptide concentration of 0.01 mg/mL at room temperature for 30 min. The quartzes or Ti screws were rinsed thoroughly with MilliQ water to remove the unbounded peptides, and then the Ti screws were immersed in 75% (v/v) ethanol for 30 min for disinfection before cell seeding or surgery. 2.4 Osteogenic differentiation of H-MSCs and OP-MSCs To induce osteoblasts, both H-MSCs and OP-MSCs were subjected to osteogenic medium， containing 10 mM β-glycerolphosphate (Sigma-Aldrich, USA), 0.1 µM dexamethasone (SigmaAldrich, St. Louis, MO, USA) and 0.25 mM ascorbate (Sigma-Aldrich,USA) in DMEM-HG (Hyclone, USA) with 10 % FBS. Some of OP-MSCs were seeded on Ti-coated glasses modified with OGP- and RGD-peptides at different ratio (0:0, 4:0, 3:1, 2:2, 0:4), respectively. Alkaline phosphatase (ALP) activity was determined by ALP staining and commercial kit (Beyotime, Nanjing, China) following the instruction of manufactory at day 7. Cells were incubated for 30 min with Alizarin Red S (Sigma-Aldrich, USA) at pH 4.1 at room temperature to evaluate calcium accumulation 14 day later. 2.5 Animals and Ovariectomy In vivo experimental studies were approved by the Animal Ethic Committees of Soochow University. All the procedures were performed followed by the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH). Three month-old female Wistar rats (with weight from 250 to 300 gram) were used in this study. The animals were


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housed in a climate-controlled environment at 25°C, 55% humidity, and 12 hours alternating light–dark cycle. Standard laboratory diet and tap water were provided ad libitum. All of the surgeries were performed under general anesthesia achieved via intraperitoneal injection of pentobarbital (30 mg/kg body weight, Merck, Germany) under sterile conditions. The animals were immobilized in the supine position, and the lower abdomen were shaved, washed, and disinfected with Anerdian. A midline longitudinal belly white line incision of lower abdomen was made, and ovariectomy was performed. 61 The animals were randomly divided into two groups: an OVX group that underwent bilateral ovariectomy to induce osteoporosis with low estrogen levels and a sham group which only the peri-ovarian fatty tissue was exteriorized. After ovariectomy, the animals were returned to their cages and were fed according to the aforementioned feeding protocol. Eight weeks after ovariectomy, just before implants surgical intervention, the OVX experimental group was scanned by in vivo micro-CT (SkyScan1176 InVivo Micro-CT, BRUKER, Kontich, Belgium) under inhalational anesthesia (Isoflurane) to assess bone quality as described later. 2.6 Screw Implantation Anesthesia and feeding methods were identical as described above. The flat lateral surface of the femoral condyle was the site for the surgical approach and screw placement. The surgical areas were shaved and disinfected by anerdian before surgical draping. The animals were immobilized in lateral position. The lateral condyle of the distal femur was exposed by an incision through the skin, fascia, and periosteum using sterile surgical techniques. First, a 1mm Kirschner wire was clipped in the low-speed electric drill, and a medullary channel drilled in the femoral condyle. The implants with or without peptides were manually screwed with a fourangle screwdriver. After insertion of the implants, the soft tissue layers and skin were closed


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with sutures. After the operation, penicillin 80,000 units per rat was administrated intraperitoneal injection for 3 continuous days, and animals were monitored daily. After completion of the implantation procedures, all the rats had free access to normal pellet food and water. 2.7 Micro-CT Analysis The bone-screw interface and trabecular micro-structure of specimens were scanned on a Micro-CT system (18µm voxel size, 65kV, 385µA, 300ms exposure time, Al 1mm filter, 180°rotation step; SkyScan1176 In-Vivo Micro-CT, BRUKER, Kontich, Belgium). Multilevel thresholding procedure (threshold for bone=135; threshold for implant=255) was applied to discriminate bone from other tissue. Before screws implantation, the volume of interest (VOI) of OVX rat was defined 1 mm from the growth plate to 3 mm trabecular of the left femoral condyles for. In the 3D VOI, the ratio between the amount of bone volume (BV) and the total volume (TV) was calculated and expressed as bone volume fraction (BV/TV). The absolute mean of trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and bone mineral density (BMD) were also determined. The connectivity of the trabecular network was characterized by the connectivity density (Conn.D). At the end of the experiment, the VOI was selected in an axisymmetric cuboid with a rectangular plane (0.9×1.8mm) from the top view and a depth of 6 mm along the longitudinal axis of the screw. The VOI included the trabecular compartment between the outer diameter and inner diameter from the longitudinal axis of the screw. Scans were reconstructed, and threedimensional digitized images were generated for each specimen using the supporting analyzing software. The BV, BV/TV, Tb.Th, Conn.D, Tb.N, and Tb.sp were calculated (CT Analyser Version 1.10; Skyscan).


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2.8 Histomorphometric Analysis To evaluate the bone response around the implants, histological and histomorphometric analyses were conducted. After formalin fixation, the implants were rinsed in water, dehydrated in ethanol, cleared in xylene, and embedded in methyl methacrylate as previously described.

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The methyl methacrylate embedding blocks were fixed in an EXAKT 300CP hard tissue slicer and cut along the longitudinal axis and the center of the implant of 300 µm of thickness produced. The slices were then placed on EXAKT 400 CS microfilm device, and grounded to 10 µm thickness. The sections were subsequently stained with toluidine blue prior to analysis. Images of the specimens were captured with a light microscope (Axio imager M1, Carl Zeiss,Germany). For the histomorphometric analysis, a computer-based image analysis system (bioquant osteo, BIOQUANT, USA) was used. Direct contact was considered between the bone matrix and the implant surface when there was no visible gaps and the bone matrix was adherent to the implant surface. The bone-to-implant contact ratio (BIC, %) was calculated at 50× magnification for three different sections per implant. Quantification was done by a blind expert operator. 2.9 Biomechanical Analysis Upon collection, all implant specimens were packaged in gauzes soaked in normal saline at 4°C until the beginning of the pull out test. The specimens were mounted on an Instron E10000, (Instron, MA, USA) equipped with a 500 N gage. A custom-made clamping system was used to adequately link the screw heads to the testing machine’s jack, and a 1 mm thick metal plate drilled with a 5 mm hole was mounted on the resin disk to stabilise the specimens. The screws were pulled out until failure using a displacement rate of 1 mm/min. The load and displacement


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were recorded. The mechanical test was stopped when complete separation of the screws from the bone and maximum load for failure were recorded. 2.10 Implant Surface Analysis The excess tissue from the screws removed from distal femoral condyle was taken away, and repeatwashing with DMSO and deionized water performed. The screws then underwent critical point drying (Autosamdri-815, Tousimis) in CO2. The surface morphology of Ti screws was imaged using a scanning electron microscope (SEM, S-4800, Hitachi). Meanwhile, the presence of calcium (Ca) and phosphorus (P) element on different zones of the Ti screws surface were semi-quantatively confirmed by energy-dispersive spectroscopy (EDS) analysis. 2.11 Statistical Analysis All data presented in this study were expressed as the mean ± standard deviation (S.D.). Statistical analyses were performed using SPSS version 14.0 for Windows software (SPSS, Chicago, IL, USA). All data were examined for normal distribution using the KolmogorovSmirnov test. One-way analysis of variance and Student–Newman–Keuls post hoc tests were used to determine the level of significance. Statistical significance was represented as *p < 0.05 and **p < 0.01. 3. Results and Discussions As mentioned above, the major reason for weak implant osseointegration in osteoporotic patients is the poor interfacial osteogenesis which caused by the impaired bone metabolism and the relatively low bioactivity of the implant surface. Therefore, osteoporosis greatly challenges the application of implant-based prosthetic rehabilitation due to the high risk of implant loosening. Based on our previous success of biomimetic peptide coatings in healthy condition,


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we anticipate that such kind of surface functionalization strategy would provide the possibility for improving the interfacial bone metabolism, osteogenicity and subsequently osseointegration in an osteoporotic condition. To this end, we developed an osteoporotic rat model for in vivo investigation. Moreover, we extracted the bone marrow cells from the osteoporotic rats for in vitro study. This would be helpful, from the points of both in vitro and in vivo, to confirm the applicability of our strategy under the condition of a disturbed bone metabolism. 3.1 Biomimetic peptide synthesis and the coating characterization The two mussel-derived bioactive peptides were prepared by standard Fmoc-based solid-phase peptide synthesis strategy.59 After HPLC purification, the two biomimetic peptides were characterized with electrospray ionization mass spectrometry (ESI-MS). The monoisotopic mass [M-H]− of (DOPA)4-S5-YGFGG were measured at 1693.1 and the monoisotopic mass [M+H]+ of (DOPA)4-S5-GRGDS and 1685.1, which were corresponding to their theoretical molecular weight 1693.68 and 1684.63, respectively. This result demonstrated the successful synthesis of two biomimetic peptides with multiple DOPA units and different bioactive motifs. Considering that, the multivalent catechol-containing sequence could be easily and stably grafted onto TiO2 surfaces through coordinative interactions, here we used TiO2-coated quartz substrates for the peptide coating and then characterized by X-ray photoelectron spectrum (XPS). As shown in Figure 1C, surface elemental compositions showed a significantly enhanced N 1s signal on peptide-coated surfaces (400.12 eV, corresponding to the amide in peptide bonds). Quantitatively, the N/Ti atomic ratio increased from 0.069 (no peptide coating) to around 0.56 (peptide coated surface) after 30 min of incubation in peptide solutions. Atom force microscope (AFM) was also used to check the surface roughness. As shown in Figure 1D-G, we can observe


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a significant surface changes after the peptide coatings. These results clearly confirmed that the two biomimetic peptide could be easily coated on the TiO2 surfaces. Insert Figure 1 3.2 Confirmation of Osteoporotic Bone Condition in Rats We developed osteoporosis in Wistar rats by ovariectomy, which is a widely used animal model of postmenopausal osteoporosis.32 To confirm osteoporosis, the bone conditions of ovariectomized (OVX group) and sham-operated rats (SHAM group) were both investigated by live animal Micro-CT analysis 8 weeks after ovariectomy. The bone mass reduction in the femoral condyles of OVX group was first confirmed by Micro-CT 3D scanning, which clearly showed up as the less trabecular bone, disorganized trabecular architecture, and expanded marrow cavities as compared to the SHAM group (Figure 2A). Micro-CT quantitative analysis further revealed in the volume of interest, the bone volume (BV), bone mineral density (BMD), mean junction density (Conn.D), numbers of trabecular bone (Tb.N), and trabecular thickness (Tb.Th) in the femoral condyles of OVX group exhibited 81%, 66%, 33% and 64% of decrease compared with that in the SHAM group, respectively. Accordingly, the trabecular space (Tb.Sp) showed more than 7-fold increase after ovariectomy for 8 weeks. (Figure. 2B). These results confirmed that osteoporosis was well-established in the ovariectomized rats. Insert Figure 2 3.3 Identification of MSCs from Osteoporotic Rats (OP-MSCs) The bone marrow-derived mesenchymal stem cells (MSCs) from healthy and osteoporotic rats were both isolated and collected. MSCs from healthy rats (H-MSCs) and osteoporotic rats (OPMSCs) both showed fibroblast-like morphology 24 h after cell isolation and formed cell colonies


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with a swirl array after 7–9 days of culture. Flow cytometry analysis showed that those cells expressed CD29 and CD90 (the stemness markers of MSCs), but did not express CD34 and CD45 antigen (hematopoietic stem cells), suggesting the success of isolation of H-MSCs and OP-MSCs (Figure S1 in the Supplementary Materials). Considering the disturbed bone metabolism of osteoporotic rats, we then checked the osteogenesis difference of the two stem cells in vitro. Upon osteogenic induction, both H-MSCs and OP-MSCs could differentiate into osteoblasts. We can clearly see the early stage osteoblast-related indicator (ALP staining) and later stage matrix mineralization (Alizarin Red S staining) (Figure 3). However, OP-MSCs exhibited significantly lower osteogenesis than H-MSCs. This may be caused by the disturbed bone metabolism equilibrium between osteogenesis and osteolysis, implying the possible reason of the poor interfacial osteogenesis on implants in osteoporotic condition. Nevertheless, this result confirmed the osteogenesis of OP-MSCs. According to this result, we then investigated in vitro osteogenesis of OP-MSCs on the peptide-modified surfaces to optimize the peptide coatings. Insert Figure 3 3.4 Optimization of the Biomimetic Peptide Coatings In Vitro. To determine the optimal ratio of OGP and RGD for peptide coatings, we seeded the OPMSCs on TiO2-coated glasses coated with OGP-peptide and RGD-peptide at different ratio (0:0, 4:0, 3:1, 2:2 and 0:4). After osteogenic induction, the osteogenesis of OP-MSCs on different coatings was assessed by detecting osteoblast-related protein expressions. Similar to our previous study on healthy MSCs, the dual-functional group with an OPG/RGD ratio at 3:1 could greatly enhance osteogenesis of OP-MSCs (Figure 4). Alkaline phosphatase (ALP) is an early marker for assessing osteoblastic metabolic activity. After 7 days of culture, all the groups with peptide


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coatings could elicit more recognizable ALP staining than the control one without peptide (Figure 4A). Moreover, quantitative results demonstrated that the dual functional group with OGP- and RGD-peptides at a ratio of 3:1 exhibited the highest alkaline phosphatase (ALP) activity as compared to the other groups (Figure 4C). It showed nearly 3-fold enhancement in ALP activity as compared to the uncoated control group. In addition, we also checked the matrix mineralization after 14 days of osteogenic induction. Matrix mineralization is a later stage indicator of osteogenesis caused by production of calcium binding proteins that can incorporate calcium ions into ECM. After staining by Alizarin Red S, we likewise found that dual-functional groups (3:1) could greatly increase matrix mineralization as compared to others (Figure 4B and 4D). OGP has been well-shown to stimulate osteoblastic lineage cells differentiation, activate alkaline phosphatase and matrix mineralization. Several signal pathways have been reported to be involved potentially in the bone formation processing, such as MAPKAPK2, ERK1/2, CREB signaling cascade in the proliferation of osteoblastic MC3T3-E1, differentiation of MSCs to osteoblasts by linking OGP to its cellular receptor, and so on 63. All the data illustrated that OGPand RGD-peptides at a ratio of 3:1 is the best ratio for OP-MSC osteogenesis on Ti-coated surface, which will be chosen for in vivo study. It is particularly worth mentioned that, although the weak osteogenesis of OP-MSCs (Figure 3), the peptide-coated group, especially the OGP and RGD dual-functional group, could greatly improve interfacial osteogenesis. We speculated that integrin-targeted RGD peptides could provide specific cell adhesion to the substrate which would enhance receptor interaction with the surface OGP-derived peptide and facilitate the shifting of disturbed bone metabolism equilibrium from osteolysis to osteogenesis. Insert Figure 4 3.5 Titanium Screws Implantation.


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After ovariectomy for 8 weeks, all the osteoporotic rats were implanted with Ti screws coated with or without the peptide coatings. In this study, four different groups, including untreated screws (denoted as PBS group), cell adhesive RGD-peptide functionalized screws (RGD group), osteogenic growth factor OGP-peptide functionalized screws (OGP group) and dual-functionalized screws (RGD+OGP group), were used for implantation. According to the above optimized results, the molar ratio of RGD and OGP in the RGD+OGP group for in vivo experiments was fixed on 1:3. The screw implanting position was precisely located in the middle of the femoral condyle but not in the knee joint cavity (Figure S2). The size of Ti screw matched perfectly with the femoral condyle of a Wistar rat, thus no mortality or femoral fractures were recorded after the implantation surgeries. Moreover, all rats were able to walk normally in 12 h after implantation and no signs of inflammation or adverse reaction were observed during the experimental period. 3.6 Micro-CT Analysis of Osseointegration After 8 weeks healing, all the rats were sacrificed under ether anesthesia and their femoral condyles with the implanted Ti screws were analyzed under Micro-CT scanner (Figure 5A-F). 64 The interfacial osteogenesis and newly formed bone condition on different peptide-treated Ti screws were evaluated. Under the same volume of interest (VOI), the dual-functional RGD+OGP group clearly exhibited the highest percentage of bone volume to tissue volume (BV/TV) (Figure 5A), which was in line with our previous results in healthy rabbits. According to our previous in vitro data, this result could also be ascribed to the improved cell adhesion and osteogenicity at the interfaces of Ti screws in RGD+OGP group. Quantitative analysis of the VOI revealed a nearly 1.23-fold increase of BV/TV in the dual-functional RGD+OGP group (8.99 ± 0.48%) as compared to the screws treated with single peptide (7.49 ± 0.61% and 7.29 ± 0.72% for OGP


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group and RGD group, respectively) and more than 1.52-fold increase compared to the PBS group (5.91 ± 1.14%). Similarly, the peri-implant trabecular microstructures in RGD+OGP group exhibited the best bone condition, which was close to that of the SHAM groups in Figure 1B. The significant interfacial osteogenicity and excellent bone condition implied that the disturbed bone metabolism on the dual-functional Ti screws was efficiently improved. For example, the values of Conn.D, Tb.N, Tb.Th in the RGD+OGP group were 71.4%, 108.7%, and 142.5 % higher than that in the untreated PBS group, and accordingly the Tb.Sp value was 94.3% lower. In fact, the RGD group and OPG group also exhibited slight improvement in bone volume and improved newly formed bone condition. However, these two groups with monobioactivity (either improving cell adhesion or inducing osteogenesis) could not realize an efficient bone formation that is comparable to the dual-functional RGD+OGP group. Moreover, no significant difference was found between the mono-bioactive RGD group and OPG group according the Micro-CT analysis. This result, from another perspective, demonstrated that the two key bioactivities on an implant (i.e., improving cell adhesion and inducing osteogenesis) are equally crucial to enhance interfacial osteogenesis and improve newly formed bone condition, in particular, in an osteoporotic condition. Insert Figure 5 3.7 Histological and Histomorphometric Examination Histological examination is a direct method to investigate the bone tissue morphology in the peri-implant area. In this work, the histological sections were stained with toluidine blue to evaluate the bone-implant contact (BIC) and bone ingrowth into the thread regions at bone/screw interfaces in osteoporotic rats.65 As shown in Figure 5G, less interfacial bone formation was observed around the untreated screws (PBS group), whereas screws in the RGD group, OGP


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group and the dual-functional RGD+OGP group displayed clearly more newly formed bone on the threads. Moreover, histological section in the RGD+OGP group revealed a contiguous bone matrix and significantly increased trabecular around the periphery of the screw, further suggesting the key role of RGD and OGP in the regulation of bone metabolism for osteogenesis. It is also worth mentioning that no multinucleated foreign body giant cells or fibrous capsules were observed in any of the screw-implanted groups. This indicated there was no inflammatory response on the bone contact interfaces. Bone–implant contact (BIC), defined as the length ratio of direct bone-to-implant contact to the total surface of the implant, is one of the criterion to quantify the degree of osseointegration. Histomorphometric analysis of the histological sections demonstrated a 1.6-fold BIC enhancement on the dual-functional Ti screws (82.4 ± 9.9% in RGD+OGP group) compared to the screws treated with single peptide (50.8 ± 9.1% and 49.2 ± 8.2% for screws in OGP and RGD groups, respectively) and more than 2.6-fold enhancement compared to the untreated screws in PBS group (31.5 ± 6.3%) (Figure 5H). The significant BIC increase in the RGD+OGP group demonstrated the excellent osseointegration of the dual-functional Ti screws in in osteoporotic condition. In-depth study on the direct comparison of BIC enhancement (versus the untreated screws) between this work and our previous work suggested that, the dualfunctionalized peptide coating strategy seems to be more efficient in osteoporosis (2.6-fold BIC enhancement) than a normal bone condition (2-fold BIC enhancement). This result is rather beyond our expectation but is similar to a previous report. Likewise, Gao and coworkers found that the immobilization of bisphosphonates on Ti implants exhibited better efficiency to prevent implant loosening in osteoporosis than that in a normal bone.47 The authors ascribed this phenomenon to the markedly activated osteoclasts in osteoporosis, which was greatly inhibited


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by bisphosphonates, thus dramatically shifting the bone metabolism equilibrium between osteogenesis and osteolysis. In our work, the significant enhancement of osseointegration in osteoporotic rats could also be related to the overwhelming change in bone metabolism equilibrium that was induced by the synergy of cell adhesive and osteogenic peptides. Although this assumption need to be further confirmed in future study, our results indicated that the dualfunctionalized peptide coating strategy is indeed feasible to improve implant osseointegration in an osteoporotic condition. 3.8 Evaluation of Bone Apposition The formation of bone-like apatite layer on the surface of implants is suggested to be very important for the direct bonding to bone tissue.66 Therefore, Energy Dispersive Spectroscopy (EDS) analysis was then performed to investigate the calcium (Ca) and phosphorus (P) composition on the surfaces of implanted screws after 8 weeks. After removal of the megascopic tissues, all the withdrawn screws were cleaned and washed repeatedly with DMSO and water. Then, we chose the convex areas of screws for EDS analysis considering that these places could efficiently preserve the surface immobilized peptides even after implantation. Each screw with 10 random areas (50×50 µm) on the convex thread was chosen for quantification of the Ca/P deposition. As shown in Table 1, the average atomic percentages of Ca and P were both higher on all the peptide-treated screws than the untreated one. In addition, the Ca/P atomic ratio in all the groups, in particular, the dual-functional OGP+RGD group, was close to the stoichiometric value of apatite.67 This result further demonstrated that the mussel-inspired peptide for dualfunctionalization of Ti screws could efficiently enhance interfacial bone apposition in the osteoporotic rats as compared to the untreated PBS group. Insert Table 1


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3.9 Biomechanical Testing It is well known that the excellent osseointegration in clinic means a stable connection between implant and the surrounding bone tissue. To finally evaluate the outcome of functional osseointegration, the anchorage force of the implanted Ti screws was measured by a previous reported biomechanical pull-out testing.68 After 8 weeks bone healing, the average maximum push-out force of Ti screws in the RGD, OGP and OGP+RGD groups were increased by 1.29fold, 1.61-fold, and 2.25-fold as compared to the PBS control group (Figure 6). The results were in line with the results of histological analysis and bone apposition, indicating the superiority of our mussel-inspired peptide coatings for the improvement of implant mechanical fixation in an osteoporotic condition. Note that the dual-functional Ti screws in OGP+RGD groups exhibited the highest enhancement (2.25-fold) in mechanical stability as compared to the untreated group. Similar to the BIC analysis, this result is also considered to be more efficient as compared to our previously work in normal bone condition (only 2.00-fold enhancement). Clearly, these ex vivo biomechanical results confirmed that our mussel-inspired bioactive peptides could efficiently enhance the osteogenicity and osseointegration of Ti implants in rats with a disturbed bone metabolism. Moreover, a dual-functionalized peptide coating strategy for the enhancement of both osteoclast adhesion and interfacial osteogenesis showed the best quality of Ti implant osseointegration. As known, Ti and its alloys are the most popular endosseous implants and osteoporosis is the most challenging condition for orthopaedic and dental implants. Therefore, the positive results in this study indicated that the highly biomimetic peptides and the facile dualfunctionalized strategy would provide a facile, safe and effective means for improving clinical outcome of Ti-based implants in the osteoporotic condition. Insert Figure 6


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4. Conclusions In summary, this work demonstrated the effectiveness of Ti screws with bioactive musselinspired peptide coatings in ovariectomized rats for improving the quantity of implant osseointegration. In vitro study indicated that the biomimetic dual-functional coating could efficiently improve the osteogenesis of osteoporosis-derived stem cells despite of their disturbed bone metabolism. Moreover, under osteoporotic in vivo condition, the dual-functional peptide coating on Ti screws could greatly enhance the interfacial osteogenesis, improve newly formed bone condition and osseointegration. Compared to our previous study in normal bone, this biomimetic dual-functional coating was found to be more efficient to improve implant osseointegration and mechanical stability in osteoporotic bone. We speculate that the significant enhancement of both osteoclast adhesion and interfacial osteogenesis was caused by the synergism of RGD and OGP peptides, which could recover regular bone metabolism equilibrium between osteogenesis and osteolysis in the osteoporotic condition. Therefore, the highly biomimetic peptides and the dual-functionalized strategy in this work would provide a facile and effective means for improving the clinical outcome of Ti-based implants in patients with a disturbed bone metabolism. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.xxxxxx. Figure S1-S2 (PDF) Conflicts of interest There are no conflicts to declare. Acknowledgements


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We gratefully acknowledge financial support from the National Natural Science Foundation of China (81772313, 21574091, 81572131, 31400826, 81301646 and 91649204), Natural Science Foundation of Jiangsu Province (BK20151210 and BK20160056), Jiangsu Provincial Special Program of Medical Science (BL2012004), Jiangsu Provincial Clinical Orthopedic Center, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References (1) Albrektsson, T.; Johansson, C. Osteoinduction, osteoconduction and osseointegration. Eur. Spine J. 2001, 10, 96-101. DOI: 10.1007/s005860100282. (2) Mavrogenis,A.F.;Dimitriou,R.;Parvizi,J.;Babis,G.C. Biology of implant osseointegration. J. Musculoskel. Neuron. 2009,9,61-71. (3) Goodman, S. B.; Yao, Z.; Keeney, M.; Yang, F. The future of biologic coatings for orthopaedic implants. Biomaterials 2013, 34, 3174-3183. DOI: 10.1016/j.biomaterials.2013.01.074 (4) Agarwal, R.; García, A. J. Biomaterial strategies for engineering implants for enhanced osseointegration and bone repair. Adv. Drug Deliver. Rev. 2015, 94, 53-62. DOI: 10.1016/j.addr.2015.03.013 (5) Goriainov, V.; Cook, R.; Latham, J. M.; Dunlop, D. G.; Oreffo, R. O. C. Bone and metal: An orthopaedic perspective on osseointegration of metals. Acta Biomater. 2014, 10, 4043-4057. DOI: 10.1016/j.actbio.2014.06.004. (6) Li, D.; Lv, P.; Fan, L.; Huang, Y.; Yang, F.; Mei, X.; Wu, D. The immobilization of antibiotic-loaded polymeric coatings on osteoarticular Ti implants for the prevention of bone infections. Biomater. Sci. 2017, 5, 2337-2346. DOI: 10.1039/c7bm00693d. (7) Geetha, M.; Singh, A. K.; Asokamani, R.; Gogia, A. K. Ti based biomaterials, the ultimate choice for orthopaedic implants–a review. Prog. Mater. Sci. 2009, 54, 397-425. DOI: 10.1016/j.pmatsci.2008.06.004. (8) Le Guéhennec, L.; Soueidan, A.; Layrolle, P.; Amouriq, Y. Surface treatments of titanium dental implants for rapid osseointegration. Dent. Mater. 2007, 23, 844-854. DOI: 10.1016/j.dental.2006.06.025. (9) Cummings, S. R.; Melton, L. J. Epidemiology and outcomes of osteoporotic fractures. Lancet 2002, 359, 1761-1767. DOI: 10.1016/S0140-6736(02)08657-9. (10) Neer, R. M.; Arnaud, C. D.; Zanchetta, J. R.; Prince, R.; Gaich, G. A.; Reginster, J. Y.; Hodsman, A. B.; Eriksen, E. F.; Ish-Shalom, S.; Genant, H. K.; Wang, O.; Mitlak, B. H. Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women


22

Page 23 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with osteoporosis. New Engl. J. Med. 2001, 344, 1434-1441. DOI ： 10.1056/NEJM200105103441904. (11) Melton, L. J.; Chrischilles, E. A.; Cooper, C.; Lane, A. W.; Riggs, B. L. How many women have osteoporosis? J. Bone Miner. Res. 2005, 20, 886-892. DOI: 10.1359/jbmr.2005.20.5.886. (12) Kanis, J. A. Diagnosis of osteoporosis and assessment of fracture risk. Lancet 2002, 359, 1929-1936. DOI: 10.1016/S0140-6736(02)08761-5. (13) Schuit, S. C. E.; Van der Klift, M.; Weel, A. E. A. M.; De Laet, C. E. D. H.; Burger, H.; Seeman, E.; Hofman, A.; Uitterlinden, A.G.; van Leeuwen, J. P. T. M.; Pols, H. A. P. Fracture incidence and association with bone mineral density in elderly men and women: the Rotterdam Study. Bone 2004, 34, 195-202. DOI: 10.1016/j.bone.2003.10.001. (14) Barbu, H. M.; Comaneanu, R. M.; Andreescu, C. F.; Mijiritsky, E.; Nita, T.; Lorean, A. Dental Implant Placement in Patients With Osteoporosis. J. Craniofac. Surg. 2015, 26, 558-559. DOI: 10.1097/SCS.0000000000001958. (15) Becker, W.; Hujoel, P. P.; Becker, B. E.; Willingham, H. Osteoporosis and implant failure: an exploratory case-control study. J. Periodontol. 2000, 71, 625-631. DOI: 10.1902/jop.2000.71.4.625 (16) Li, Y.; He, S.; Hua, Y., Hu, J. Effect of osteoporosis on fixation of osseointegrated implants in rats. J. Biomed. Mater. Res. B 2016, 105, 2426-2432. DOI: 10.1002/jbm.b.33787. (17) Tsolaki, I. N.; Madianos, P. N.; Vrotsos, J. A. Outcomes of Dental Implants in Osteoporotic Patients. A Literature Review. J. Prosthodont. 2009, 18, 309-323. DOI: 10.1111/j.1532849X.2008.00433.x. (18) Gittens, R. A.; Olivares-Navarrete, R.; Schwartz, Z.; Boyan, B. D. Implant osseointegration and the role of microroughness and nanostructures: lessons for spine implants. Acta Biomater. 2014, 10, 3363-3371. DOI: 10.1016/j.actbio.2014.03.037. (19) Abuhussein, H.; Pagni, G.; Rebaudi, A.; Wang, H. L. The effect of thread pattern upon implant osseointegration. Clin. Oral Implan. Res. 2010, 21, 129-136. DOI: 10.1111/j.16000501.2009.01800.x. (20) Zhang, W.; Wang, G.; Liu, Y.; Zhao, X.; Zou, D.; Zhu, C.; Jin, Y.; Huang, Q.; Sun, J.; Liu, X.; Jiang, X.; Zreiqat, H. The synergistic effect of hierarchical micro/nano-topography and bioactive ions for enhanced osseointegration. Biomaterials 2013, 34, 3184-3195. DOI: 10.1016/j.biomaterials.2013.01.008. (21) Raines, A. L.; Olivares-Navarrete, R.; Wieland, M.; Cochran, D. L.; Schwartz, Z.; Boyan, B. D. Regulation of angiogenesis during osseointegration by titanium surface microstructure and energy. Biomaterials 2010, 31, 4909-4917. DOI: 10.1016/j.biomaterials.2010.02.071. (22) Steigenga, J.; Al-Shammari, K.; Misch, C.; Nociti Jr, F. H.; Wang, H. Effects of implant thread geometry on percentage of osseointegration and resistance to reverse torque in the tibia of rabbits. J. Periodont. 2004, 75, 1233-1241. DOI: 10.1902/jop.2004.75.9.1233. (23) Shibli, J. A.; Grassi, S.; de Figueiredo, L. C.; Feres, M.; Marcantonio, E. J.; Iezzi, G.; Piattelli, A. Influence of implant surface topography on early osseointegration: a histological study in human jaws. J. Biomed. Mater. Res. B 2007, 80, 377-385. DOI: 10.1002/jbm.b.30608.


23


Page 24 of 37

(24) Rosenbaum, J.; Versace, D. L.; Abbad-Andallousi, S.; Pires, R.; Azevedo, C.; Cenedese, P.; Dubot, P. Antibacterial properties of nanostructured Cu-TiO2 surfaces for dental implants. Biomater. Sci. 2017, 5, 455-462. DOI: 10.1039/c6bm00868b. (25) Frenkel, S. R.; Simon, J.; Alexander, H.; Dennis, M.; Ricci, J. L. Osseointegration on metallic implant surfaces: effects of microgeometry and growth factor treatment. J. Biomed. Mater. Res. 2002, 63, 706-713. DOI: 10.1002/jbm.10408. (26) Ripamonti, U.; Roden, L. C.; Renton, L. F. Osteoinductive hydroxyapatite-coated titanium implants. Biomaterials 2012, 33, 3813-3823. DOI: 10.1016/j.biomaterials.2012. 01.050 (27) Lakstein, D.; Kopelovitch, W.; Barkay, Z.; Bahaa, M.; Hendel, D.; Eliaz, N. Enhanced osseointegration of grit-blasted, NaOH-treated and electrochemically hydroxyapatite-coated Ti– 6Al–4V implants in rabbits. Acta Biomater. 2009, 5, 2258-2269. DOI: 10.1016/j.actbio.2009.01.033 (28) Alghamdi, H. S.; Bosco, R.; van den Beucken, J. J.; Walboomers, X. F.; Jansen, J. A. Osteogenicity of titanium implants coated with calcium phosphate or collagen type-I in osteoporotic rats. Biomaterials 2013, 34, 3747-3757. DOI: 10.1016/j.biomaterials.2013. 02.033. (29) Jimbo, R.; Coelho, P. G.; Vandeweghe, S.; Schwartz-Filho, H. O.; Hayashi, M.; Ono, D.; Andersson, M.; Wennerberg, A. Histological and three-dimensional evaluation of osseointegration to nanostructured calcium phosphate-coated implants. Acta Biomater. 2011, 7, 4229-4234. DOI: 10.1016/j.actbio.2011.07.017 (30) Schwarz, M. L.; Kowarsch, M.; Rose, S.; Becker, K.; Lenz, T.; Jani, L. Effect of surface roughness, porosity, and a resorbable calcium phosphate coating on osseointegration of titanium in a minipig model. J. Biomed. Mater. Res. A 2009, 89, 667-678. (31) Leon, B.; Jansen, J. A. Thin calcium phosphate coatings for medical implants. New York, Springer; 2009, vol 309. (32) Agarwal, R.; González-García, C.; Torstrick, B.; Guldberg, R. E.; Salmerón-Sánchez, M.; García, A. J. Simple coating with fibronectin fragment enhances stainless steel screw osseointegration in healthy and osteoporotic rats. Biomaterials 2015, 63, 137-145. DOI: 10.1016/j.biomaterials.2015.06.025 (33) Liu, L.; Tian, X.; Ma, Y.; Duan, Y.; Zhao, X.; Pan, G. A Versatile Mussel-Inspired Dynamic Biointerface: From Specific Cell Behavior Modulation to Selective Cell Isolation. Angew. Chem. Int. Ed. 2018, in press, DOI: 10.1002/anie.201804802. (34) Hennessy, K. M.; Clem, W. C.; Phipps, M. C.; Sawyer, A. A.; Shaikh, F. M.; Bellis,S.L. The effect of RGD peptides on osseointegration of hydroxyapatite biomaterials. Biomaterials 2008,29,3075-3083. DOI: 10.1016/j.biomaterials.2008.04.014. (35) Petrie, T. A.; Raynor, J. E.; Reyes, C. D.; Burns, K. L.; Collard, D. M.; García, A. J. The effect of integrin-specific bioactive coatings on tissue healing and implant osseointegration. Biomaterials 2008,29,2849-2857. DOI: 10.1016/j.biomaterials.2008.03.036. (36) Guo, B.; Pan, G.; Guo, Q.; Zhu, C.; Cui, W.; Li, B.; Yang, H. Saccharides and temperature dual-responsive hydrogel layers for harvesting cell sheets. Chem. Commun. 2015, 51, 644-647. DOI: 10.1039/c4cc08183h.


24

Page 25 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(37) Uchida, M.; Oyane, A.; Kim, H. M.; Kokubo, T.; Ito, A. Biomimetic Coating of Laminin– Apatite Composite on Titanium Metal and Its Excellent Cell‐Adhesive Properties. Adv. Mater. 2004, 16, 1071-1074. DOI: 10.1002/adma.200400152. (38) Petrie,T.A.; Reyes,C.D.; Burns,K.L.; García,A.J. Simple application of fibronectin–mimetic coating enhances osseointegration of titanium implants. J. Cell. Mol. Med. 2009, 13, 2602-2612. Doi: 10.1111/j.1582-4934.2008.00476.x. (39) Jimbo, R.; Sawase, T.; Shibata, Y.; Hirata, K.; Hishikawa, Y.; Tanaka, Y.; Bessho, K.; Ikeda, T.; Atsuta, M. Enhanced osseointegration by the chemotactic activity of plasma fibronectin for cellular fibronectin positive cells. Biomaterials 2007, 28, 3469-3477. DOI: 10.1016/j.biomaterials.2007.04.029. (40) Ren, X.; Wu, Y.; Cheng, Y.; Ma, H.; Wei, S. Fibronectin and bone morphogenetic protein2-decorated poly (OEGMA-r-HEMA) brushes promote osseointegration of titanium surfaces. Langmuir 2011, 27, 12069-12073. DOI: 10.1021/la202438u. (41) Stadlinger, B.; Pilling, E.; Huhle, M.; Mai, R.; Bierbaum, S.; Bernhardt, R.; Scharnweber, B.; Kuhlisch, E.; Hempel, U.; Eckelt, U. Influence of extracellular matrix coatings on implant stability and osseointegration: an animal study. J. Biomed. Mater. Res. B 2007, 83, 222-231. DOI: 10.1002/jbm.b.30787. (42) Guillot, R.; Gilde, F.; Becquart, P.; Sailhan, F.; Lapeyrere, A.; Logeart-Avramoglou, D.; Picart, C. The stability of BMP loaded polyelectrolyte multilayer coatings on titanium. Biomaterials. 2013, 34, 5737-5746. DOI: 10.1016/j.biomaterials.2013.03.067. (43) Liu, Y.; Enggist, L.; Kuffer, A. F.; Buser, D.; Hunziker, E. B. The influence of BMP-2 and its mode of delivery on the osteoconductivity of implant surfaces during the early phase of osseointegration. Biomaterials 2007, 28, 2677-2686. DOI: 10.1016/j.biomaterials.2007.02.003. (44) Shi, Z.; Neoh, K.; Kang, E.; Poh, C. K.; Wang, W. Surface functionalization of titanium with carboxymethyl chitosan and immobilized bone morphogenetic protein-2 for enhanced osseointegration. Biomacromolecules 2009, 10, 1603-1611. DOI: 10.1021/bm900203w. (45) López-Lacomba, J. L.; García-Cantalejo, J. M.; Sanz Casado, J. V.; Abarrategi, A.; Correas Magaña, V.; Ramos, V. Use of rhBMP-2 Activated Chitosan Films To Improve Osseointegration. Biomacromolecules 2006, 7, 792-798. DOI: 10.1021/bm050859e. (46) Alghamdi, H. S.; Bosco, R.; Both, S. K.; Iafisco, M.; Leeuwenburgh, S. C. G.; Jansen, J. A.; van den Beucken, J. J. Synergistic effects of bisphosphonate and calcium phosphate nanoparticles on peri-implant bone responses in osteoporotic rats. Biomaterials 2014, 35, 54825490. DOI: 10.1016/j.biomaterials.2014.03.069. (47) Gao, Y.; Zou, S.; Liu, X.; Bao, C.; Hu, J. The effect of surface immobilized bisphosphonates on the fixation of hydroxyapatite-coated titanium implants in ovariectomized rats. Biomaterials 2009, 30, 1790-1796. DOI: 10.1016/j.biomaterials.2008.12.025. (48) Du, Z.; Chen, J.; Yan, F.; Xiao, Y. Effects of Simvastatin on bone healing around titanium implants in osteoporotic rats. Clin. Oral Implant. Res. 2009, 20, 145-50. DOI: 10.1111/j.16000501.2008.01630.x.


25


Page 26 of 37

(49) Andersen, O. Z.; Offermanns, V.; Sillassen, M.; Almtoft, K. P.; Andersen, I. H.; Sørensen, S.; Jeppesen, C. S.; Kraft, D. C.; Bøttiger, J.; Rasse, M.; Kloss, F.; Foss, M. Accelerated bone ingrowth by local delivery of strontium from surface functionalized titanium implants. Biomaterials 2013, 34, 5883-5890. DOI: 10.1016/j.biomaterials.2013.04.031. (50) Offermanns, V.; Andersen, O. Z.; Riede, G.; Andersen, I. H.; Almtoft, K. P.; Sørensen, S.; Sillassen, M.; Jeppesen, C. S.; Rasse, M.; Foss, M.; Kloss, F., Bone regenerating effect of surface-functionalized titanium implants with sustained-release characteristics of strontium in ovariectomized rats. Int. J. Nanomed. 2016, 11, 2431-2442. DOI: 10.2147/IJN.S101673。 (51) Li, Y.; Xiong, W.; Zhang, C.; Gao, B.; Guan, H.; Cheng, H.; Li, F. Enhanced osseointegration and antibacterial action of zinc‐loaded titania‐nanotube‐coated titanium substrates: In vitro and in vivo studies. J. Biomed. Mater. Res. A 2014, 102, 3939-3950. DOI: 10.1002/jbm.a.35060. (52) Zeng. X.; Liu, G.; Tao, W.; Ma, Y.; Zhang, X.; He, F.; Pan, J.; Mei, L.; Pan, G. A DrugSelf-Gated Mesoporous Antitumor Nanoplatform Based on Ph-Sensitive Dynamic Covalent Bond. Adv. Funct. Mater. 2017, 27, 1605985. DOI: 10.1002/adfm.201605985. (53) Pan, G.; Guo, Q.; Ma, Y.; Yang, H.; Li, B. Thermo‐Responsive Hydrogel Layers Imprinted with RGDS Peptide: A System for Harvesting Cell Sheets. Angew. Chem. Int. Ed. 2013, 52, 6907-6911. DOI: 10.1002/anie.201300733. (54) Meyers, S. R.; Grinstaff, M. W. Biocompatible and Bioactive Surface Modifications for Prolonged In Vivo Efficacy. Chem. Rev. 2012, 112, 1615-1632. Doi: 10.1021/cr2000916. (55) Pan, G.; Shinde, S.; Yeung, S.; Jakštaitė, M.; Li, Q.; Wingren, A. G.; Sellergren, B. An Epitope-Imprinted Biointerface with Dynamic Bioactivity for Modulating Cell–Biomaterial Interactions. Angew. Chem. Int. Ed. 2017, 56, 15959-15963. DOI: 10.1002/anie.201708635. (56) 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, DOI: 10.1016/j.biomaterials.2016.01.016. (57) Pan, G.; Guo, B.; Ma, Y.; Cui, W.; He, F.; Li, B.; Yang, H.; Shea, K. J. Dynamic introduction of cell adhesive factor via reversible multicovalent phenylboronic acid/cis-diol polymeric complexes. J. Am. Chem. Soc. 2014, 136, 6203-6206. DOI: 10.1021/ja501664f. (58) Bab, I.; Gazit, D.; Chorev, M.; Muhlrad, A.; Shteyer, A.; Greenberg, Z.; Namdar, M.; Kahn, A. Histone H4-related osteogenic growth peptide (OGP): a novel circulating stimulator of osteoblastic activity. EMBO J. 1992, 11, 1867-1873. (59) Pan, G.; Sun, S.; Zhang, W.; Zhao, R.; Cui, W.; He, F.; Huang, L.; Lee, S. H.; Shea, K. J.; Shi, Q.; Yang, H. Biomimetic Design of Mussel-Derived Bioactive Peptides for DualFunctionalization of Titanium-Based Biomaterials. J. Am. Chem. Soc. 2016, 138, 15078-15086. DOI: 10.1021/jacs.6b09770. (60) Batul, R.; Tamanna, T.; Khaliq, A.; Yu, A. Recent progress in the biomedical applications of polydopamine nanostructures. Biomater. Sci. 2017, 5, 1204-1229. DOI: 10.1039/c7bm00187h. (61) Kalu, D. N. The ovariectomized rat model of postmenopausal bone loss. Bone and Mineral 1991, 15, 175-191. DOI: 10.1016/0169-6009(91)90124-I.


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(62) Liu, S. Chen, H.; Wu, T.; Pan, G.; Fan, C.; Xu, Y.; Cui, W. Macrophage infiltration of electrospun polyester fibers. Biomater. Sci. 2017, 5, 1579-1587. DOI: 10.1039/c6bm00958a. (63) Pigossi, S. C.; Medeiros, M. C.; Saska, S.; Cirelli, J. A.; Scarel-Caminaga, R. M. Role of Osteogenic Growth Peptide (OGP) and OGP(10-14) in Bone Regeneration: A Review. Int. J. Mol. Sci. 2016, 17, PII:e1885. DOI: 10.3390/ijms17111885. (64) Jones, A. C.; Milthorpe, B.; Averdunk, H.; Limaye, A.; Senden, T. J.; Sakellariou, A.; Sheppard, A. P.; Sok, R. M.; Knackstedt, M. A.; Brandwood, A.; Rohner, D.; Hutmacher, D. W. Analysis of 3D bone ingrowth into polymer scaffolds via micro-computed tomography imaging. Biomaterials 2004, 25, 4947-4954, DOI: 10.1016/j.biomaterials.2004.01.047. (65) Park, J. W.; Kurashima, K.; Tustusmi, Y.; An, C. H.; Suh, J. Y.; Doi, H.; Nomura, N.; Noda K.; Hanawa, T. Bone healing of commercial oral implants with RGD immobilization through electrodeposited poly(ethylene glycol) in rabbit cancellous bone. Acta Biomater. 2011, 7, 32223229. DOI: 10.1016/j.actbio.2011.04.015. (66) Jonášová, L.; Müller, F. A.; Helebrant, A.; Strnad J.; Greil, P. Biomimetic apatite formation on chemically treated titanium. Biomaterials 2004, 25, 1187-1194. DOI: 10.1016/j.biomaterials.2003.08.009 (67) Raynaud, S.; Champion, E.; Bernache-Assollant D.; Thomas, P. Calcium phosphate apatites with variable Ca/P atomic ratio I. Synthesis, characterisation and thermal stability of powders. Biomaterials 2002, 23, 1065-1072. DOI: 10.1016/S0142-9612(01)00218-6. (68) Aparicio, C.; Padrós A.; Gil, F. J. In vivo evaluation of micro-rough and bioactive titanium dental implants using histometry and pull-out tests. J. Mech. Behav. Biomed. Mater. 2011, 4, 1672-1682. DOI: 10.1016/j.jmbbm.2011.05.005.


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Figure Captions: Scheme 1. Chemical structures of the biomimetic peptides with multiply catechol units and bioactive peptides on their chains. The peptides can spontaneously bind on Ti screw via strong multivalent coordination interactions, and then the functionalized Ti screw was implanted in an osteoporotic rat to check the effect on osseointegration. Figure 1. ESI mass spectrum (DOPA)4-S5-GRGDS (A) and (DOPA)4-S5-YGFGG (B). (C) X-ray photoelectron spectrum (XPS) of the bare Ti surface and modified Ti surfaces after 2 h of incubation in mussel-derived peptides solutions (0.01 mg·mL–1 in PBS, 25 °C). Atom force microscope (AFM) images of the bare (D), (DOPA)4-S5-GRGDS coated (E), (DOPA)4-S5-YGFGG coated (F) and dual-peptide coated (1:1) (G) Ti screw surfaces. Figure 2. A) Representative micro-CT images of femoral condyles in OVX group and SHAM group. B) Quantitative results of the bone morphological alterations including bone volume (BV), bone mineral density (BMD), mean junction density (Conn.D), numbers of trabecular bone (Tb.N), trabecular thickness (Tb.Th) and trabecular space (Tb.Sp) in the region of interest (n=6). Star (*) indicates significant difference between columns (*p < 0.05, ** p < 0.01). Figure 3. In vitro osteogenesis of BMSCs derived from healthy rat and osteoporosis rat. A) Representative images of ALP staining and quantitative ALP activity of the BMSCs after 7 days of culture in osteogenic induction medium. Scale bar is 200 µm. B) Representative images and quantification of Alizarin red staining of the BMSCs after 14 days of culture in osteogenic induction medium. Scale bar is 100 µm. (*p < 0.05, ** p < 0.01). Figure 4. In vitro osteogenesis of the OP-BMSCs on the biomimetic peptide-treated TiO2 surfaces. (a) Representative images of ALP staining after 7 days of culture in osteogenic induction medium. (b) Representative images of Alizarin Red S staining after 14 days of culture in osteogenic induction medium. (c) Quantitative ALP activity after 7 days of culture. (d) Quantification of the Alizarin Red S stained mineral layer after 14 days of culture. TiO2-coated substrates were incubated in PBS with different ratios of OGP peptide and RGD peptide (i.e., 0:0, 4:0, 3:1, 2:2, and 0:4, total concentration was 0.01 mg·mL−1) for 6 h at room temperature. Scale bar is 200 µm. (*p < 0.05, ** p < 0.01). Figure 5. A) Micro-CT 3D reconstructed models showing the status of the Ti implant (red in color) and the response of bone (white in color) at 8 weeks after implantation. B-F) Micro-CT analysis of BV/TV, Tb.Th, Conn.D, Tb.N, and Tb.sp (n=6). G) Representative histological sections stained with toluidine at 8-week after implantation. Insets show the magnified images in red boxes. H) Histomorphometrical results of bone-implant contact (BIC) (n=4). (*p < 0.05, **p < 0.01). Figure 6. A) Biomechanical pull-out testing curves and B) the average pull-out strength of different peptidetreated and untreated Ti screws (n=5). Star (*) indicates significant difference between columns (*p < 0.05, **p < 0.01). Table.1 EDS semi-quantitative elemental analysis of the calcium (Ca) and phosphorus (P) composition on the implanted Ti screws.


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Scheme 1. Chemical structures of the biomimetic peptides with multiply catechol units and bioactive peptides on their chains. The peptides can spontaneously bind on Ti screw via strong multivalent coordination interactions, and then the functionalized Ti screw was implanted in an osteoporotic rat to check the effect on osseointegration.


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Figure 1. ESI mass spectrum (DOPA)4-S5-GRGDS (A) and (DOPA)4-S5-YGFGG (B). (C) X-ray photoelectron spectrum (XPS) of the bare Ti surface and modified Ti surfaces after 2 h of incubation in mussel-derived peptides solutions (0.01 mg·mL–1 in PBS, 25 °C). Atom force microscope (AFM) images of the bare (D), (DOPA)4-S5-GRGDS coated (E), (DOPA)4-S5-YGFGG coated (F) and dual-peptide coated (1:1) (G) Ti screw surfaces.


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Figure 2. A) Representative micro-CT images of femoral condyles in OVX group and SHAM group. B) Quantitative results of the bone morphological alterations including bone volume (BV), bone mineral density (BMD), mean junction density (Conn.D), numbers of trabecular bone (Tb.N), trabecular thickness (Tb.Th) and trabecular space (Tb.Sp) in the region of interest (n=6). Star (*) indicates significant difference between columns (*p < 0.05, ** p < 0.01).


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Figure 3. In vitro osteogenesis of BMSCs derived from healthy rat and osteoporosis rat. A) Representative images of ALP staining and quantitative ALP activity of the BMSCs after 7 days of culture in osteogenic induction medium. Scale bar is 200 µm. B) Representative images and quantification of Alizarin red staining of the BMSCs after 14 days of culture in osteogenic induction medium. Scale bar is 100 µm. (*p < 0.05, ** p < 0.01).


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Figure 4. In vitro osteogenesis of the OP-BMSCs on the biomimetic peptide-treated TiO2 surfaces. (a) Representative images of ALP staining after 7 days of culture in osteogenic induction medium. (b) Representative images of Alizarin Red S staining after 14 days of culture in osteogenic induction medium. (c) Quantitative ALP activity after 7 days of culture. (d) Quantification of the Alizarin Red S stained mineral layer after 14 days of culture. TiO2-coated substrates were incubated in PBS with different ratios of OGP peptide and RGD peptide (i.e., 0:0, 4:0, 3:1, 2:2, and 0:4, total concentration was 0.01 mg·mL−1) for 6 h at room temperature. Scale bar is 200 µm. (*p < 0.05, ** p < 0.01).


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Figure 5. A) Micro-CT 3D reconstructed models showing the status of the Ti implant (red in color) and the response of bone (white in color) at 8 weeks after implantation. B-F) Micro-CT analysis of BV/TV, Tb.Th, Conn.D, Tb.N, and Tb.sp (n=6). G) Representative histological sections stained with toluidine at 8-week after implantation. Insets show the magnified images in red boxes. H) Histomorphometrical results of bone-implant contact (BIC) (n=4). (*p < 0.05, **p < 0.01).


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Figure 6. A) Biomechanical pull-out testing curves and B) the average pull-out strength of different peptidetreated and untreated Ti screws (n=5). Star (*) indicates significant difference between columns (*p < 0.05, **p < 0.01).


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Table.1 EDS semi-quantitative elemental analysis of the calcium (Ca) and phosphorus (P) composition on the implanted Ti screws. Group

n

Ca（（atom %））

P（（atom %））

Ca/P

PBS

18

0.87±0.45

0.79±0.39

1.10

RGD

18

1.48±0.98

1.26±0.87

1.17


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For Table of Contents Use Only

Mussel-Inspired Peptide Coatings on Titanium Implant to Improve Osseointegration in Osteoporotic Condition Huan Zhao, Yingkang Huang, Wen Zhang, Qianping Guo, Wenguo Cui, Zhiyong Sun, David Eglin, Lei Liu, Guoqing Pan,* Qin Shi *


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Mussel-Inspired Peptide Coatings on Titanium Implant to Improve

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