3D-Printed Bioactive Ca3SiO5 Bone Cement Scaffolds with Nano

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3D-printed bioactive Ca3SiO5 bone cement scaffolds with nano surface structure for bone regeneration Chen Yang, Xiaoya Wang, Bing Ma, Haibo Zhu, Zhiguang Huan, Nan Ma, Chengtie Wu, and Jiang Chang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14297 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on January 27, 2017

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ACS Applied Materials & Interfaces

3D-printed bioactive Ca3SiO5 bone cement scaffolds with nano surface structure for bone regeneration Chen Yang †, Xiaoya Wang †, Bing Ma †, Haibo Zhu ‡, Zhiguang Huan †, Nan Ma ‡, Chengtie Wu †, and Jiang Chang*† †State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, China. ‡ Xuhui District Central Hospital, 966Middle Huaihai Road, Shanghai, 200031, China.

Keywords: 3D printing, tricalcium silicate cement, scaffold, drug-loading, nanotopography, osteogenesis, bone formation

ACS Paragon Plus Environment

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Abstract

Silicate bioactive materials have been widely studied for bone regeneration because of their eminent physicochemical properties and outstanding osteogenic bioactivity, and different methods have been developed to prepare porous silicate bioactive ceramics scaffolds for bone tissue engineering applications. Among all these methods, 3D-printing technique is obviously the most efficient way to control the porous structure. However, 3D printed bioceramic porous scaffolds need high temperature sintering which will cause volume shrinkage and reduce the controllability of the pore structure accuracy. Unlike Silicate bioceramic, bioactive silicate cements such as tricalcium silicate (Ca3SiO5, C3S) can be self-set in water to obtain high mechanical strength under mild conditions. Another advantage of using C3S to prepare 3D scaffolds is the possibility of simultaneous drug loading. Herein, we, for the first time, demonstrated successful preparation of uniform 3D-printed C3S bone cement scaffolds with controllable 3D structure at room temperature. The scaffolds were loaded with two model drugs and showed loading location controllable drug release profile. In addition, we developed a surface modification process to create controllable nanotopography on the surface of pore wall of the scaffolds, which showed activity to enhance rat bone marrow stem cells (rBMSCs) attachment, spreading and ALP activities. The in vivo experiments revealed that the 3D-printed C3S bone cement scaffolds with nanoneedle structured surface significantly improved bone regeneration as compared to pure C3S bone cement scaffolds, suggesting that 3D-printed C3S bone cement scaffolds with controllable nanotopography surface are bioactive implantable biomaterials for bone repair.


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1. Introduction In recent years, there has been a significant increase in the demand for bone graft materials. Many synthetic biomaterials including Si-containing bioactive ceramics have been considered as new bone-regenerative materials due to their distinct properties such as excellent mechanical properties, high apatite inducing activity, controllable degradation rate, and outstanding osteogenic and angiogenic bioactivity in vitro and in vivo.1-5 Considering these significant advantages, many methods including polyurethane foam templating and porogens techniques have been used to prepare Si-containing bioactive ceramics as porous scaffolds for bone tissue engineering applications.6-7 However, the pore size and morphology of the scaffolds prepared by these methods are difficult to control, which directly influence scaffolds’ interconnection, mechanical strength and in vivo bone-formation ability, further limits their clinical applications. To overcome these limitations, 3D printing techniques have been applied to prepare porous scaffolds in recent years. Three dimension structures can be created layer-by-layer as designed by using injected paste or polymer.8-9 The advantages of 3D printed scaffolds are the controllable pore morphology, pore size and porosity as compared to that prepared by conventional methods.10 Many bioceramics including calcium phosphate and Si-containing bioceramics have been fabricated as three dimensional porous scaffolds by 3D printing technique.3, 11 Yet, all these scaffolds need high temperature sintering after printing which is not only energy consuming, but also hard to control the pore structures because of the volume shrinkage during sintering process. Also, high temperature sintering makes scaffolds impossible for spatially localized drug or growth factor incorporation which restricts their biomedical applications. It would be idea if we can find a silicate material, which can be 3D printed at room temperature to form stable scaffolds without requirement of further high temperature sintering after printing. Unlike Si-containing


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bioceramics, bioactive silicate cements (another important type of Si-containing bioactive materials) possess hydraulic property and spontaneous development of strength in aqueous environment under mild conditions.12-13 Tricalcium silicate (Ca3SiO5, C3S), one of the bioactive silicate cements has been used in endodontic treatment, and recent in vitro and in vivo studies have demonstrated that C3S is bioactive and biocompatible, and has potential for bone regeneration applications.12, 14 Therefore, we assume that C3S may be the idea materials for 3D printing of stable silicate macroporous scaffolds at room temperature without further sintering. The combination of C3S cement and 3D printing technique will make the whole manufacturing process ‘green’ and economic. Also, this low temperature rapid prototyping of C3S cement offers advantages over sintering routes in that organic molecules, such as drugs or growth factors may be selectively incorporated in the printing process throughout an entire graft for specific applications.15 For bone tissue engineering, an optimally designed scaffold should be capable of stimulating the bone regeneration process.16-17 The role of structural design of porous scaffolds is one of the vital factors in stimulating and guiding the bone regeneration process. Many studies have shown that surface nano-topography could affect the stem cell fate, enhance osteogenic differentiation of stem cells, and ultimately promote new bone formation.18-23 To obtain nanostructured surface on bioceramics, many strategies have been used including hydrothermal treatment and simulated body fluids (SBF) immersion.24-27 Our previous study also revealed that different nano structured hydroxyapatite (HAp) structure can be obtained through high temperature hydrothermal process, in which the silicate bioceramic functions as a hard template,28-29 and this kind of nano structure is able to stimulate osteogenesis of stem cells and bone regeneration.30 However, there is still no


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report on the creation of nano-structure on the surface of C3S bone cements in particular under low temperature condition. Herein, we combined 3D printing technique with a mild process to fabricate 3D macroporous C3S bone cement scaffolds with different nano surface structure, and investigated their effect on rat bone marrow stem cells growth, osteogenic differentiation and bone regeneration in vivo.

2. Materials and methods 2.1. Synthesis of C3S powders C3S powders were prepared by sol–gel method according to a previous report,31 for which Ca(NO3)2·4H2O and Si(OC2H5)4 (TEOS) were used as the raw materials and nitric acid was used as catalyst. Briefly, 1.5mol Ca(NO3)2·4H2O was added to the solution containing 0.5 mol TEOS and 200 ml water under continuous stirring. After Ca(NO3)2·4H2O was completely dissolved, the mixture was kept to be stirred for another 1 h. Subsequently, the solution was kept at 60 °C for 24 h to complete gelation. The gels was then dried at 120 °C for 12 h, and followed by the sintering at 1450 °C for 8 hours. The calcinated powders were ground in a milling ball and sieved to obtain the powders with a maximal powder size of 38 µm. 2.2. 3D printing of C3S bone cement scaffolds Hydroxypropylmethylcellulose (HPMC) was selected as the binder because it is a biocompatible, biodegradable, hydrophilic polymer with desirable mechanical properties.32-33 In a typical experiment, 4 g of C3S powders were mixed with 1.8 g of an aqueous HPMC solution (1.5 wt.%) for printing. The 3D printing equipment used in this study was developed by Fraunhofer IWS (Dresden, Germany) and has an accurate three-axis positioning system (GeSiM,


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Grosserkmannsdorf, Germany) for controlling the size and pore parameters of the scaffolds. The dosing pressure to the syringe pump was 200–400 kPa and the speed of the dispensing unit was 10 mm s-1. In this study, different macropore structures are designed for in vitro cell culture and in vivo animal experiments, respectively. Up and down staggered scaffolds were printed for vitro study to facilitate effective cell seeding on materials,34 while traditional up and down connected scaffolds were printed for in vivo study, which the interconnected pore networks and may be more suitable for bone formation.35-36 2.3. Self-setting and characterization of C3S bone cement scaffolds The C3S bone cement scaffolds obtained were self-set in deionized water at 37 °C for 0, 1, 3, 7and 14 days. The crystal phase of C3S bone cement scaffolds were characterized by X-ray diffraction (XRD) (D8ADVANCE, Bruker, Germany). The overall morphologies of C3S bone cement scaffolds were observed using an optical Nikon D90 camera. The macropore structure and micro or nano morphology of the pore walls were investigated by scanning electron microscopy (SEM) (S-4800, Hitachi, Japan). The porosity was determined in compliance with the water displacement method.37 Briefly, the weights of the dried scaffolds, water-filled scaffolds, buoyant scaffolds (immersed in water) were recorded as M1, M2 and M3, respectively. The porosity (P) was calculated using the equation: P= (M2-M1)/ (M2-M3) × 100%. 2.4. In vitro study of drug loading and release properties To investigate the flexibility of drug loading in 3D printed C3S bone cement scaffolds, two model drugs, rhodamine B (RHB) and calcein (purchased from Sinopharm Chem), were loaded.


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Briefly, 10 mg RHB and 5mg calcein were dissolved in 1g HPMC aqueous solution respectively. Drug loaded scaffolds were printed by the same method described in 2.2 under a designed program to make scaffolds with half RHB-loading and half calcein-loading. In the simple model experiment, 0.4 g scaffold sample with half RHB and half calcein loading were horizontally placed in a big container with 40 mL of phosphate buffer solution (PBS) at pH 7.4. One testing point was close to RHB-loading part (Fig 4(b) A) and the other testing point was close to calcein-loading part (Fig 4(b) B). At the predetermined time intervals, 150µL solutions were taken out to centrifuge and 100 µL solutions were tested, and all the solutions were replaced with fresh PBS. The concentrations of both RHB (at an optical wavelength of 554nm) and calcein (at an optical wavelength of 484 nm) were determined using a microplate spectrophotometer, respectively. The concentration was calculated based on the standard curve and the data were expressed as mean ± standard deviation (n=5). 2.5. Fabrication and characterization of C3S bone cement scaffolds with nanoneedle and nanosheet surface structure A modified method was applied to fabricate the 3D printed C3S bone cement scaffolds with nanoneedle or nanosheet surface structure by transforming C3S to nano-hydroxyapatite at 37 °C in different phosphate aqueous solution.29,

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Briefly, 0.3 M Na2HPO4 and (NH4)2HPO4

aqueous solution were prepared respectively at room temperature. Then, 1g C3S bone cement scaffolds were immersed in Na2HPO4 and (NH4)2HPO4 aqueous solution at 37 °C for 3 days with a volume of 100 mL. Finally, all these scaffolds were soaked in deionized water at 37 °C for 4 days. To further confirming the phase of formed nano-structured surfaces, pulverized powders of C3S scaffolds were treated in the same phosphate solution at 37 °C with an excessive


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volume for 7 days. The phase of the bulk samples and powders was determined by using X-ray diffraction analysis (D8ADVANCE, Bruker, Germany). The surface morphology and the fracture morphology of the samples was observed by scanning electron microscopy (SEM) (S4800, Hitachi, Japan). 2.6. Mechanical strength of C3S bone cement scaffolds The C3S bone cement scaffolds for compressive strength test were prepared in a size of 10×10×10 mm, and the samples were immersed in deionized water for 0, 1, 3, 7and 14 days at 37 °C before the test using a computer-controlled universal testing machine (AG-I, Shimadzu, Japan) at a crosshead speed of 0.5 mm min-1. Also, the compressive strength of C3S bone cement scaffolds with nanostructured surfaces were tested compared to pure C3S bone cement scaffolds. Five samples were tested for each time point. 2.7. The effect of nanostructured surface on in vitro degradation of C3S bone cement scaffolds For evaluation of degradation, Tris–HCl solution with pH value of 7.4 was chosen as testing buffer considering to mimic the in vivo environment which may close to neutral condition. Scaffold samples were immersed in Tris–HCl solution (1g sample/200ml solution) at 37 °C in a shaking bath for up to 28 days, and the solution was refreshed every three days. Then, the samples were dried at 60°C for 24 h after the set soaking time. The weight loss of each sample was calculated based on the weight measurement. 2.8. The effect of nanostructured surfaces on cell attachment and proliferation


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Rat bone marrow stem cells (rBMSCs) (obtained from Allcells, Silicon Valley, CA, USA) were seeded with an initial density of 1× 104 on each scaffold in 48-well tissue culture plates containing 500µl Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS),100 U/ml penicillin and 100 µg/ml streptomycin. The cellseeded scaffolds were cultivated in an incubator at 37 °C and 5% CO2, and the cell culture medium was changed every 3 days. The images of actin cytoskeletons labeling were taken by the confocal laser scanning microscopy (Leica, Wetzlar, Germany). Briefly, cells were cultured on the scaffolds for 6 hours, and then were washed with PBS and fixed in 4% paraformaldehyde for 30 min followed by wash with PBS for two times. The actin cytoskeletons were stained with Phalloidia-TRITC (Sigma) for 30 minutes and the cell nuclei was contrast-labeled using 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) for 5 minutes. The morphology of the cells were visualized using a confocal laser scanning microscope (Leica, Wetzlar, Germany). In addition, the scaffolds were prepared for SEM observing, in which the samples after culturing for 1 day were fixed in 2.5% glutaraldehyde overnight at 4 °C. After that, the scaffolds were rinsed with PBS for three times and then dehydrated in a graded series of ethanol (20%, 30%, 50%, 70%, 95%,100%), 5 min each. Finally, the samples were dried by hexamethyldisilazane, sputter-coated with gold and examined by SEM (S-4800, Hitachi, Japan) to observe the adhesion and growth of rBMSCs on C3S bone cement scaffolds. The cell proliferation was measured by using MTT assay which was performed as follows: 500 µL culture medium containing 0.5 mg/mL of MTT dye was added to each sample and maintaining for 4h. After removal of the MTT solution, 300 µL of DMSO (dimethylsulfoxide) was added and the formazan extract was quantified by measuring the absorbance at 590 nm


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using a microplate reader (Epoch microplate spectrophotometer, Bio Tek, USA). The data of the cell proliferation were obtained by deducting the absorbance value of blank wells from the overall optical density values. 2.9. The effect of nanostructured surfaces on ALP activity of RBMSCs After 7 days culturing, the ALP activity of RBMSCs seeded on scaffolds was measured. Briefly, the cultured cells were detached from the scaffolds by trypsin digestion, and the detached cells were rinsed with PBS for two times, followed by centrifugation for 5 min at 1000 rpm, and then resuspended in lysis buffer with 0.2% p-nitrophenol (pNP). Each sample was evenly mixed with 1 mg mL1 p-nitrophenyl-phosphate (pNPP) in 1 M diethanolamine buffer and incubated at 37 °C for 30 min. The ALP activity was quantified by reading the absorbance at 405 nm according to a series of p-nitrophenol (pNP) standards. The Bradford method was applied to determine the total protein content in aliquots of the same samples using the Bio-Rad protein assay kit (BioRad, Richmond, USA). The total protein content was measured at the absorbance of 630 nm and counted in accordance with the BSA standards, and the ALP activity of the cells was shown as absorbance at 405 nm (OD value) per milligram of total cellular proteins. To determine the ion release behavior of the scaffolds, the cell culture medium for ALP activity test was collected, and the ionic concentrations of Ca, Si and P were measured using an inductively coupled plasma atomic emission spectrometry (ICP-AES, VarianCo., USA). 2.10. The scaffold implantation into femur defects of rabbits. All experiments were carried out by following the relevant laws and guidelines. The C3S (without spatial nanostructured surfaces) bone cement scaffolds and C3S-NN (with nanoneedle surfaces) bone cement scaffolds with a size of Ø6 × 10 mm were implanted into femoral bone


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defects of adult New Zealand rabbits (twelve rabbits with defects created both for right and left posterior limbs, Experimental Animal Center of Shanghai No.1 Medical University, Shanghai, China) by surgery. An intravenous injection of 20% urethane (4 mL kg−1) was performed for general anesthesia. Then, a 6.0 mm drill was used to create critical size defects in femur with a size of 6 mm in diameter and 10 mm in depth. Then, one C3S and one C3S-NN scaffold were implanted into the defect of the right and left posterior limbs in one rabbit, respectively. Afterward, every four rabbits were sacrificed at 1 month, 3 months and 6 months. Then all samples were removed from rabbits for an X-ray (X’Pert PRO, Panalytical, Netherlands) before other treatment. 2.11. Histological and histomorphometric observation To obtain paraffin-embedded tissue samples, 10% EDTA was used as the decalcifying agent, and the samples were soaked in the solution and refreshed every two weeks. The whole procedure last for 5 weeks. Paraffin sections were cut at a thickness of approximately 4 µm and fixed on polylysine-coated microscope slides. The slides were stained using hematoxylin and eosin (H&E), and were observed using a visual light microscopy (S2500, Leica, Germany) in order to generally assess the tissue and wound healing. The newly formed bone was quantitatively evaluated using a personal computer-based image analysis system (Image Pro 6.0, Media Cybernetic, USA) and presented as percentage of the total area of defects. 2.12. Statistical analysis The data are expressed as means ± standard deviations (SD). One-way ANOVA with a post hoc test was used by establishing the statistical significance, and a p value lower than 0.05 was considered as statistical significance.


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3. Results 3.1. Characterization of 3D printed C3S bone cement scaffolds 3D printed C3S bone cement scaffolds were printed in different shapes (Fig 1a), and the pores were highly interconnected as shown by the top view images (Fig 1b) and side-view image (Fig 1c). SEM analysis revealed that C3S bone cement scaffolds have controllable macroporous morphologies (Fig 2). The line width and pore size could be also controlled under 200 µm (Fig 2a–c). The porosity of C3S bone cement scaffolds could be designed by using different preparation procedures (Fig 2a-f). Neither toxic solvents nor high temperature was used in the process.

Fig 1. Overview of C3S bone cement scaffolds. (a) 3D-printed C3S bone cement scaffolds with different sizes and shapes. (b) Top view of C3S bone cement scaffolds. (c) Side view of C3S bone cement scaffolds.


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Fig 2. SEM images of C3S bone cement scaffolds on top view (a, d), bottom view (b, e) and side view (c, f). 3.2 Self-setting of 3D printed C3S bone cement scaffolds The strut surface morphology of scaffold is are shown in Fig 3a-f after maintained in deionized water for various times (1d, 3d, 7d and 14d). It showed that the sample was porous and tiny particles aggregated on the surface (Fig 3b-d) in early time. With the increase of the hydration time, these particles developed into fibre-like structure and the surface became more dense (Fig 3e-f). XRD patterns of C3S bone cement scaffolds before and after soaking in deionized water for various times (1d, 3d, 7d and 14d) at 37 °C were shown in Fig 3g. Only tricalcium silicate peaks were discernible for the C3S bone cement scaffold before its setting. Over time, a remarkable change can be seen in XRD patterns, and new peaks of calcium silicate hydrate (CSH) phase (JCPDS card: No. 03-0548) and calcium hydroxide (Ca(OH)2) phase (JCPDS card: No. 040733) appeared, which are typical C3S hydration products. Significantly, hydration reaction of


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C3S seems to be completed after 7 days curing, as no obvious changes in the XRD pattern from day 7 to 14 was observed.

Fig 3. SEM images of C3S bone cement scaffolds after soaking in deionized water for various times. (a) Micrographs of the surfaces of scaffolds. (b, c, d, e, f) The high magnification images of the macropore walls after soaking in deionized water for 0, 1, 3, 7and 14 days, respectively. (g) XRD patterns of C3S bone cement scaffolds before and after setting in deionized water for various times. 3.3. Drug-loading and release properties of 3D printed C3S bone cement scaffolds in vitro


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By applying 3d printing technique, different drugs can be precisely loaded in different location of scaffolds, which may result in different drug concentrations for different drugs in different area. In this work, scaffolds with half RHB-loading and half calcein-loading (Fig 4a) were used as a simple model to show the controlled drug loading in 3D scaffolds and the release profile. The drug release profiles at two different locations (as indicated in Fig 4b as A and B) near the due-drug loaded scaffolds were measured, one was close to RHB-loading part and the other was close to calcein-loading part respectively. It is clear to see that, in the near of RHB loaded part of the scaffold (point A), the RHB concentration was significantly higher than that of the calcein for the first 24 hours (Fig 4c), while in the near of calcein loaded part of the scaffold the calcein concentration was significantly higher than that of RHB (Fig 4d) indicating a controllable specific drug concentration is achievable by controlling the drug loading location during 3D printing process.


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Fig 4. Drug loading in 3D printed C3S scaffolds. (a) An image of dual-drug loaded scaffolds with RHB in one part (dark area) and calcein in another part (light area) of the scaffolds. (b) Illustration of two measuring point of drug concentrations for dual-drug release up to 48 h from co-drug loaded C3S cement scaffolds. (c) Concentration of RHB measured at point A. (d) Concentration of calcein measured at point B. 3.4. Creation of nanostructured surface on 3D printed C3S bone cement scaffolds Considering the bioactive effect of nano hydroxyapatite structure on bone regeneration, we designed a process to treat the 3D printed C3S bone cement scaffolds in order to obtain nanostructured hydroxyapatite on the surface of scaffolds by immersing them in specific phosphate aqueous solution at 37℃ for 3 day. The results showed that nanoneedle and nanosheet structures were obtained on the surface at 37℃ comparing with scaffolds immersed in deionized water (Fig 5). Especially, the nanoneedle structures can be obtained in 0.3M Na2HPO4 aqueous solution and nanosheet structures can be obtained in 0.3M (NH4)2HPO4 aqueous solution. Fig 5b and Fig 5c show that the formed nanoneedle and nanosheet structures were homogeneous and covered the strut surface of the scaffolds. SEM images with higher magnification show that low temperature treated scaffold consisted of nanoneedles (C3S-NN) with diameters about 20 nm and lengths up to 300nm (Fig 5e), or consisted of nanosheets (C3S-NS) with thickness of 8 nm, widths of 500 nm, and lengths up to 200 nm (Fig 5f), while as expected, the control sample (Fig 5a, d) showed no special micro or nanostructures on the scaffolds’ surface. Images of the fracture surface (Fig 5h, i) showed that the nano-structure topographies were only formed on the surface of the struts.


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Fig 5. SEM images of C3S bone cement scaffolds after soaking in deionized water (a, d, g), Na2HPO4 aqueous solution (b, e, h) and (NH4)2HPO4 aqueous solution (c, f, i) at 37℃ for 3 days. The surface images (a-f) showed that the nanoneedle structures (b, e) and nanosheet structures (c, f) were formed on the surface of the scaffolds after treating with phosphate aqueous solution comparing with of scaffolds (a, d) immersed in deionized water. Cross-section images (g-i) further confirmed that only a small amount of nano-structures formed on the surface of the scaffolds. Figure 6a shows the XRD patterns of nanoneedle (C3S-NN) and nanosheet (C3S-NS) samples after low temperature treatment in Na2HPO4 and (NH4)2HPO4 aqueous solution. The results clearly revealed that hydration reaction of C3S was slower in phosphate solution as compared to that in deionized water. The main phase of C3S-NN and C3S-NS scaffolds is obviously still C3S or CSH, and only small amount of hydroxyapatite crystals was formed on the


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surface of these samples. The phases of the hydroxyapatite were further confirmed by XRD analysis (Figure 6b) of treated C3S powders pulverized from scaffolds in the same phosphate solution.

Fig 6. XRD patterns of C3S bone cement scaffolds (a) after soaking in deionized water (C3S), Na2HPO4 aqueous solution (C3S-NN) and (NH4)2HPO4 aqueous solution (C3S-NS) at 37℃ for 3 days. XRD patterns of pulverized powders of C3S scaffolds (b) after soaking in excessive phosphate solution for 7 days.


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3.5. Mechanical strength of 3D printed C3S bone cement scaffolds The results of mechanical and porosity tests of the scaffolds with different curing time (0d, 1d, 3d, 7d and 14d) are shown in Fig 7. The results showed that the compressive strength and modulus of 3D printed C3S bone cement scaffolds increased with time, which reached 12.9 Mpa (Fig 7a) and 680.9 Mpa (Fig 7b) with porosity of 61% (Fig 7c) after setting for 7 days, respectively. The compressive strength of 3D printed C3S bone cement scaffolds with nanostructured surfaces were also evaluated, and no significant difference was observed between C3S, C3S-NN and C3S-NS scaffolds after curing for 7days (Fig 7d).

Fig 7. The relationship between compressive property and porosity. (a)The compressive strength, (b) The Young’s modulus and (c) The porosity of C3S bone cement scaffolds after soaking in deionized water for 0, 1, 3, 7and 14 days. (d)The compression strength of C3S bone cement scaffolds with and without nanostructured surfaces.


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3.6. The effect of nanostructured surfaces in vitro degradation of 3D printed C3S bone cement scaffolds In vitro degradation of the scaffolds was evaluated by measuring weight loss after soaking them in Tris–HCl solution. The result of the weight loss shows a sustained weight loss with time proceeding (Fig 8). The weight loss of C3S, C3S-NN and C3S-NS scaffolds was 24.2%, 18.3%, 20.2%, respectively, after soaking for 28 days. Obviously, nanostructured surfaces reduced in vitro degradation of C3S bone cement, and C3S-NN scaffolds showed lowest weight loss.

Fig 8. The weight loss of C3S bone cement scaffolds with or without nanostructure after soaking in Tris–HCl solution at 37℃ for 28 days. 3.7. The effect of nanostructured surfaces on the attachment, proliferation and ALP activity of rBMSCs The early attachment of rBMSCs after seeding on C3S, C3S-NN and C3S-NS scaffolds for 6h was observed by actin cytoskeletons labeling (Fig 9). The fluorescence images showed that cells spreading out better on C3S-NN (Fig 9b, e, h) and C3S-NS (Fig 9c, f, i) scaffolds as compared with those on C3S scaffolds (Fig 9a, d, g) without nanostructure”


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Fig 9. Confocal images of rBMSC cultured on C3S (a, d, g), C3S-NN (b, e, h) and C3S-NS (c, f, i) for 6 hours. Scale bar = 25 µm.

Fig 10. SEM images of rBMSC cultured on C3S (a, d, g), C3S-NN (b, e, h) and C3S-NS (c, f, i) for 1 day.


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The SEM observation showed that rBMSCs attached and spread well on all the scaffolds at day 1 (Fig 10). There was no significate difference between C3S scaffolds with and without nano surface structure.

Fig 11. RBMSC proliferation (a) and ALP activity (b) on C3S, C3S-NN and C3S-NS bone cement scaffolds. The amount of released (c) Ca, (d) P and (e) Si of C3S, C3S-NN and C3S-NS bone cement scaffolds after cells culture for ALP activities test. MTT results showed that the proliferation of RBMSCs was obviously increased on C3S, C3S-NN and C3S-NS scaffolds with the increase of culture time (Fig 11a). However, there is no significant difference among between different groups. In contrast, cells on both the C3S-NN and C3S-NS scaffolds had significant higher ALP activities than C3S scaffolds, and C3S-NN scaffolds possessed the highest ALP activity after culturing for 7 days (Fig 11b). The ionic concentrations for Ca, P and Si of medium after cell culture for ALP activities test are shown in Fig 11c-e. The concentration of Ca and Si ions released from C3S scaffolds was significantly higher than that of C3S-NN or C3S-NS scaffolds. There is no significant difference of Ca or P


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concentration between C3S-NN and C3S-NS scaffold groups. However Si ions released from C3S-NN scaffolds (about 22 ppm) was higher than that of C3S-NS scaffolds (about 8 ppm). 3.8. The in vivo bone formation of C3S bone cement scaffolds C3S-NN scaffolds were selected for in vivo study to assess the in vivo bone formation as compare to the pure C3S scaffolds, due to their higher activity to induce osteogenic differentiation observed in cell culture experiments. Photo images of surgery before (Fig 12a) and after (Fig 12b) implantation of the scaffolds is shown in Fig 12.

Fig 12. Images of surgery before (a) and after (b) scaffolds were transplanted into femur defects. X-rays images of the bone defect repair with C3S scaffolds (Fig 13a-c) and C3S-NN scaffolds (Fig 13d-f) after implantation for various time periods are shown in Fig 13. Obviously, all scaffolds integrated well with the host bone tissue and degraded with time.


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Fig 13. X-rays of C3S scaffolds (a-c) and C3S-NN scaffolds (d-f) after implanted for 1 month (a, d), 3 months (b, e) and 6 months (c, f).

Fig 14. Evaluation of in vivo bone formation after implantation by means of H&E staining. C3S scaffolds (a-c) and C3S-NN scaffolds (d-f) after implanted for 1 month (a, d), 3 months (b,e) and 6 months(c, f), quantitative analysis (g) for new bone formation. NB: new bone; M: materials. Scale bar = 200 µm. H&E staining showed that the quantity of new bone increased apparently over time throughout the implantation for both C3S and C3S-NN scaffolds (Fig 14) while the new bone formed in C3S-NN implantation group was significantly more than that in the C3S scaffolds implantation group. One month after implantation, new bone tissue started to grow into C3S-NN scaffolds (Fig 14d), while there was limited new bone tissue growing into C3S scaffolds (Fig 14a). Six months after implantation, macropores of C3S-NN scaffolds were almost fully filled with newly formed bone (Fig 14f). By the contrast, it was seen that the new bone formation in


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the defects filled with C3S scaffolds was rather limited, which was mostly located around the macropores (Fig 14c). The quantitative results of the new bone formation are shown in Fig 14g, which revealed that more new bone formed in C3S-NN scaffolds as compared to that in C3S scaffolds.

4. Discussion Recently, silicate bioactive ceramics have attracted a lot of attention for the application in bone tissue regeneration due to their excellent bioactivity.39-42 Many silicate bioactive ceramic scaffolds have been explored for enhancing bone regeneration via different methods in the past two decades.6-7 Until now, significant progress has been made in preparation of macroporous structure of ceramic scaffolds by applying 3D printing technique,3,

11

which can efficiently

control the internal parameters of the porous structure such as pore size, morphoplogy and distribution, and the external geometry of the scaffolds. However, all these ceramic scaffolds need high temperature sintering after printing process, which may result in unavoidable volume shrinkage and affect the final structure of the scaffolds. Also, high temperature sintering limits the applications of 3D printing in combination with spatially localized drug or growth factor incorporation. Unlike silicate bioceramics, bioactive silicate cements such as tricalcium silicate have self-setting property and can solidify under mild conditions. This unique property makes it possible to be 3D printed at room temperature to form macroporous scaffolds with controllable structure without further sintering. And under this mile condition, spatially localized drug/growth factor loading for specific applications is also possible. Here, for the first time, we reported successful fabrication of C3S cement scaffolds at 37 oC by using 3D printing method.


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C3S cements possess hydraulic property to form a solid network composed of CSH and Ca(OH)2 with increasing curing time, which is associated with the densification and decrease of the porosity due to the development of microstructure during the hydration reaction, and the decrease of the porosity resutls an increase of the strength.12-13 This process is quite slow, which makes it more suitable for 3D printing process as compared to other type of inorganic cement materials such as calcium phosphate cements, which have been used for 3D printing of calcium phosphate scaffolds.8,

43

The quick setting time of conventional CPCs may have significant

impact on the homogeneity and mechanical properties of the scaffolds,15, 43-45 while the slow hydration property of C3S results in a slow setting process and allows a more smooth printing process with controllable macropore morphologies, pore sizes and porosities (Fig 1-2), and a higher mechanical strength may also be obtained. The minimum line diameter and pore size of 3D printed C3S bone cement scaffolds could be controlled to nearly 170 µm, which is obviously more sophisticated than reported 3D printed CPC scaffolds.8, 10, 43, 45-46 Furthermore, the maximal compressive strength of 3D printed C3S bone cement scaffolds obtained in our study reached 12.9 Mpa with porosity of 61%, which is much higher than that of 3D printed CPC scaffolds reported in literature. (0.9-8.7 Mpa with the porosity in the range of 41-64%).43-45 The other obvious advantage of self-setting materials is the in situ drug loading property.47-48 Drugs can be loaded to 3D printed CPC scaffolds during 3D printing process.49 One advantage of 3D printing silicate cement scaffolds is the spatially localized drug loading during the printing process, and in particular, two or more different drugs can be loaded within one scaffold in different spatial distribution of different drugs, which may be used for specific applications. For example, for the regeneration of articular cartilage and subchondral bone, bilayer scaffolds may be applied, and it will be idea to have the upper layer releasing growth factors for cartilage


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regeneration, while the lower layer releases growth factors for bone regeneration. Here, we used a two drug model by loading RHB and calcein in two different parts of one scaffold and demonstrated that individual drugs can be loaded precisely in the wanted part of the scaffold and the release profile confirmed a concentration differentiation of two drugs with a higher drug concentration of one drug on one side of the scaffold and the other higher on another side. In our previous study, we have fabricated bioceramics with nanoneedle and nanosheet hydroxyapatite (HAp) surface structure by using hydrothermal treatment of the ceramic scaffolds at high temperature, and demonstrated that these nano surface structures could enhance bone regeneration.27, 50 It is meaningful to modify the 3D printed C3S cement scaffolds with nanostructured HAp in particular at low temperature. According to our previous experience, hydroxyapatite with nanoneedle structure was easier to be obtained under higher pH condition, and lower pH value contributed to nanosheet structure.38 In this study, we successfully achieved hydroxyapatite nanostructured surface on 3D printed C3S bone cement scaffolds by transforming tricalcium silicate in different phosphate aqueous at 37 oC. Here, we choose Na2HPO4 aqueous solution and (NH4)2HPO4 aqueous solution to control the formation of different nano structure, and nanoneedle structure was obtained in Na2HPO4 solution, while nanosheet structure was obtained mainly in (NH4)2HPO4. This is the first report for creation controllable HAp nanoneedle and nanosheet structure on C3S scaffolds at body temperature. The bioactive effect of nano-structured surface was confirmed by culturing rBMSCs on the 3D printed C3S bone cement scaffolds with nanoneedle (C3S-NN) and nanosheet (C3S-NS) structures. The results show that the initially adherent cell number on C3S-NN and C3S-NS scaffolds was higher and cells presenting a better spreading as compared with those on C3S scaffolds without nanostructure, and the ALP activity of rBMSCs on both the C3S-NN and C3S-


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NS scaffolds was also significant higher than that on C3S scaffolds without nanostructure, and C3S-NN scaffolds revealed the highest ALP activity. In addition to the effect of nano-surface structure, which has been confirmed to affect osteogenic differentiation of stem cells18, 27, 50 one possible reason for the highest bioactivity of C3S-NN might be the release of bioactive ions. It is known that Si plays a vital role in stimulating osteogenic differentiation of cells and promoting bone new formation in vivo.3-4 However, an overdose of Si may have negative effect on cells and too low Si concentration may not have bioactivity.40, 51 Therefore, it is critical to control the Si ion concentration in the proper range. In the present study, the Ca and Si concentration released from scaffolds were significantly reduced due to the formation of nano-structured HAp surface, as well as the increased P concentration indicating the role of surface modification on the control of ion release. The results show that the Si concentration from C3S-NN scaffolds group is about 22 ppm, which is moderate as compared to C3S and C3S-NS. Our results suggest that C3S-NN have the best combination of nanostructure and bioactive ions for stimulating cell differentiation. Considering the higher activity of nanoneedle surface structure, C3S-NN scaffolds were chosen to evaluate the in vivo effect of the nanostructured surface for bone regeneration, and the in vivo results further demonstrated the excellent activity of the C3S cement scaffolds with nanoneedle surface structure on bone regeneration. These results confirmed our previous finding with similar nanostructure on bioceramic scaffolds, and suggested that 3D printed silicate cement scaffolds with nanostructure modified surface are good candidate for bone regeneration applications.

5. Conclusion In this study, highly uniform C3S bone cement scaffolds were successfully fabricated using 3D-printing technique for the first time. The obtained 3D printed C3S bone cement scaffolds


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have excellent pore structure and high mechanical strength, and different drugs could be easily loaded in different location of the scaffolds, which resulted in different drug release profile in different direction. Furthermore, the 3D printed C3S cement scaffolds were surface modified by creation of nanostructured topographies via a crystal growth process with C3S as the precursor under a mild conditions. The C3S cement scaffolds with nanostructured topographies slowed activity to promoted rBMSCs attachment, spreading and osteogenic differentiation with the nanoneedle structure showed the highest activity. The in vivo study demonstrated that the 3Dprinted C3S bone cement scaffolds with nanoneedle surface structure significantly enhanced bone regeneration as compared to the pure C3S bone cement scaffolds. Our results suggest that the 3D printing silicate bone cements, with the advantages of low temperature fabrication and controllable multiple and defined spatial drug loading function, is a novel approach for preparing bone tissue engineering, and 3D printed C3S bone cement scaffolds with controllable nanotopography surface may be used for bone regeneration applications.

Author information Corresponding Author *E-mail: [email protected] (J.C.), Tel./Fax: 86-21-52412804. Notes The authors declare no competing financial interest.

Acknowledgements


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Funding for this study was provided by National Key Research and Development Program of China (2016YFB0700803), Natural Science Foundation of China (Grant 81430012, 81190132, 81671830, 31370963), Key Research Program of Frontier Sciences ， CAS (QYZDB-SSWSYS027) and Program of Shanghai Outstanding Academic Leaders (15XD1503900).

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3D-Printed Bioactive Ca3SiO5 Bone Cement Scaffolds with Nano

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