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Article
3D printing of hollow struts-packed
bioceramic scaffolds for bone regeneration
Yongxiang Luo, Dong Zhai, Zhiguang Huan, Haibo Zhu, Lunguo Xia, Jiang Chang, and Chengtie Wu
ACS Appl. Mater. Interfaces, Just Accepted Manuscript DOI: 10.1021/acsami.5b08911 Publication Date (Web): 19 Oct 2015
Downloaded from http://pubs.acs.org on October 22, 2015
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Page 1 of 29 ACS Applied Materials & Interfaces
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6 3D printing of hollow struts-packed bioceramic scaffolds for bone
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9 regeneration
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13 a,b
14 Yongxiang Luo , Dong Zhai a, Zhiguang Huan a, Haibo Zhu c, Lunguo Xia d, Jiang
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16 Chang a*, Chengtie Wu a*
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21 a
State Key Laboratory of High Performance Ceramics and Superfine Microstructure,
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24 Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050,
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26 Peoples Republic of China.
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b
29 Department of Biomedical Engineering, Health Science Center, Shenzhen University,
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31 Shenzhen 518060, P.R. China
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c
34 Xuhui District Central Hospital, 966 Middle Huaihai Road, Shanghai, China
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36 d
Center of Craniofacial Orthodontics, Department of Oral and Cranio-maxillofacial
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39 Science, Ninth Peoples Hospital Affiliated to Shanghai Jiao Tong University, School
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of Medicine, Shanghai 200011, China
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44 * Corresponding authors
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E-mail: jchang@mail.sic.ac.cn (J. Chang); Tel +86-21-52412804, Fax
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49 +86-21-52413903
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E-mail: chengtiewu@mail.sic.ac.cn (C. Wu); Tel +86-21-52412249, Fax
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54 +86-21-52413903
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ACS Paragon Plus Environment
ACS Applied Materials & Interfaces Page 2 of 29
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4 Abstract:
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6 3D printing technologies have shown distinct advantages to create porous scaffolds
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9 with designed macro pores for the application in bone tissue engineering. However,
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11 until now, 3D printed bioceramic scaffolds only owning single type of macropores
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14 have been reported. Generally, those scaffolds with single type of macropores had
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16 relative low porosity and pore surfaces, and limited delivery of oxygen and nutrition
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19 for cells surviving, new bone tissue formation in the center of scaffolds. Therefore, in
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21 this work, we presented a useful and facile method for preparing hollow struts packed
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24 (HSP) bioceramics scaffolds with designed macropores and multi-oriented hollow
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26 channels via a modified coaxial 3D printing strategy. The prepared HSP scaffolds
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29 combined high porosity and surface area with impressive mechanical strength. The
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31 unique hollow-struts structures of bioceramic scaffolds significantly improved cell
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34 attachment and proliferation, and further promoted formation of new bone tissue in
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36 the center of scaffolds, indicating that HSP ceramic scaffolds can be used for
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39 regeneration of large bone defects. In addition, the strategy can be used for prepare
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other HSP ceramic scaffolds, indicating a universal application for tissue engineering,
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44 mechanical engineering, catalysis and environment materials.
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48 Keywords: 3D printing, bioceramics, hollow-struts scaffolds, bone tissue engineering
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4 1. Introduction
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6 Treatment of critical-size bone defects caused by trauma, infection and osteoporosis
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9 remains significant clinical challenges.1 An expected approach is to implant synthetic
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11 porous scaffolds into defects for guiding and stimulating formation of new bone
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14 tissues.2 However, scaffold-based bone tissue engineering is still unable to effectively
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16 overcome some problems, such as poor host tissue integration and insufficient bone
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19 formation in the inner of large bone defects due to the restrictions of homogeneous
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21 cells distribution, migration and exchange of oxygen and nutrition in the inner of
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24 scaffolds.3,4 To address these issues, it is of great importance to design smart scaffolds
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26 with the ability to guide and stimulate formation of new bone tissue in large bone
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29 defects.5-8 It is known that the nature of biomaterials plays a crucial role for satisfying
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31 this requirement,9,10 in which the biological activity of scaffolds not only depends on
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34 material components, but also the macroporous architecture. Previous studies
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36 demonstrated that the geometrical features of porous scaffolds, including surface
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39 curvature, pore shape and size had significant impact on cellular response and in vivo
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bone regeneration.11-14 Although modulating the geometrical features of scaffolds can
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44 partly improve their bone-forming ability, the strategy is far from optimal to
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effectively solve the long-term existing issue of insufficient bone formation in the
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49 inner of large-size scaffolds. Several previous studies tried to solve these problems by
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creating hollow channels in scaffolds via wire array template15,16 and sacrificial
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54 modeling17,18 strategies. In this process, porous silk scaffolds with mono-oriented
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hollow channels were prepared by freeze drying in combination of wire array
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4 template. The prepared hollow channels could facilitate cells infiltration. However,
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6 the methods were limited for preparing polymer-based scaffolds with mono-oriented
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9 hollow channels. In addition, the macropores in the scaffolds are uncontrollable by
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11 using freeze-drying method.
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14 Herein, we reported a facile method to fabricate hollow struts-packed (HSP)
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16 bioceramic scaffolds with designed macro pores and multi-oriented hollow channel
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19 structures via co-axis 3D printing in order to enhance the bone regeneration. Such
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21 novel scaffolds have significantly improved porosity and surface area for cells
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24 migration and new bone formation.
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26 2. Materials and methods
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29 2.1 3D printing hollow struts-packed (HSP) bioceramic scaffolds
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31 Bioceramics (Ca7Si2P2O16) powders were synthesized by a sol-gel process using
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34 tet-raethyl orthosilicate ((C2H5O)4Si), triethyl phosphate ((C2H5O)3PO) and calcium
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36 nitrate tetrahydrate (Ca(NO3)24H2O) according to a previous study.19 The received
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39 powders were grinded to particle size less than 38 m by sieving to 400 mesh. For
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preparing the printable bioceramic ink (paste), 5 g of bioceramic powders were mixed
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44 with 0.3 g of alginate powder (Alfa Aesar) and 2.8 g of Pluronic F-127 (20 wt.%)
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(Sigma Aldrich) aqueous solution then stirred until homogeneous pastes were
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49 achieved. HSP bioceramic scaffolds were fabricated by printing the prepared pastes
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through design of a shell/core nozzle in printing system (as shown in Figure 1a). The
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54 3D printing system was developed by Fraunhofer IWS (Dresden, Germany) based on
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the Nano-Plotter device from GeSiM (Grosserkmannsdorf, Germany) and introduced
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4 on the market as a bio-scaffold printer.20 Solid struts-packed (SSP) bioceramic
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6 scaffolds were printed based on the similar process only via signal nozzles and used
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9 for controls. For controlling the porosity of scaffolds, several size of shell/core
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11 nozzles were designed and prepared for this study, including those with a standard of
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14 (shell/core nozzle, G): 16/21, 16/22, 16/23, 18/25, 20/27. After scaffolds were printed,
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16 they were dried over night under room temperature and then sintered at 1400C for 3
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19 h.
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21 2.2 Characterization of the printed HSP bioceramic scaffolds
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24 The macropore and hollow channel morphology of the sintered scaffolds were
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26 observed by optical microscopy (S6D, Leica, Germany). The macropores and
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29 microstructure of the pore walls were characterized by scanning electron microscopy
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31 (SEM) (JSM-6700F, JEOL, Japan). The porosity was measured according to a liquid
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34 displacement method. 21 In brief, the scaffolds were first dried at 100 C overnight and
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36 weighed, and marked as M1. Then the scaffolds were immersed in water and placed
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39 under vacuum until no bubbles appeared. The weight of the scaffolds with water-filled
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pores was marked as M2. Finally, the scaffolds were immersed in water and the buoyant
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44 weight was marked as M3. The porosity (P) was calculated using the equation:
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P=( M 2 -M 1 )/ (M 2 -M 3 )1 00% .
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2.3 Mechanical testing and in vitro degradation
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54 The compressive strength and modulus of the obtained scaffolds (101010 mm)
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with different porosity (controlled by macropores and hollow channels) were tested
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4 using a computer controlled universal testing machine (AG-I, Shimadzu, Japan) at a
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6 cross-head speed of 0.5 mm min-1. Five samples were tested for each type of scaffold.
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9 For comparing the degradation of HSP and SSP Scaffolds, two kinds of scaffolds
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11 (printed with a nozzle of core/shell: 16/22) were soaked in TrisHCl buffered solution
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14 (pH 7.40) in a shaking water bath at 37 for 7, 14, 21 and 28 days. The solution was
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16 refreshed every 7 days. The ratio of solution volume to scaffold mass was 50 mL g-1.
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19 Three samples were used for repeat experiments. At each time point the weight loss
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21 was calculated. To investigate ionic release from the scaffolds the solution was
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24 collected every 7 days. The concentrations of Ca, P and Si ions were determined by
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26 inductively coupled plasma atomic emission spectrometry (ICP-AES) (Varian Co.,
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29 USA).
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31 2.4 Cell culture in vitro
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34 Human BMSCs (obtained from Cambrex, Walkersville, MD, USA) were seeded with
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36 an initial density of 5104 on each scaffold. The cell-seeded scaffolds were cultivated
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39 in an incubator at 37 and 5% CO2, and the cell culture medium was changed twice
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per week.
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44 MTT assay was given after 1, 3 and 7 days of cultivation to analysis the proliferation
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of the cells on scaffolds. Briefly, 1 mL of the MTT solution (0.5 mg mL-1 in cell
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49 culture medium) was added to each sample. After incubation for 4 h, the solution was
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removed and 300 L of DMSO (dimethyl sulfoxide) was added to solubilize the
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54 formazan product. An aliquot of 100l was transferred to a fresh 96-well plate;
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absorbance was measured at 590 nm in a microplate reader (Epoch microplate
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4 spectrophotometer, Bio Tek Instruments, USA). All the data are presented as optical
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6 density values minus the absorbance of blank wells. For preparing cells-scaffolds
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9 samples for SEM and confocal LSM analysis, after washing with phosphate buffered
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11 saline (PBS) and fixed with 2.5% glutaraldehyde, they were dried in
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14 hexamethyldisilizane (HMDS) for 30 min. Then, samples were coated with carbon
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16 and stained with Rhodamine Phalloidin-DAPI, and observed by SEM (FEI Magellan
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19 400) and confocal microscopy (Leica TCS SP8), respectively.
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21 2.5 In vivo evaluation
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24 All experiments were performed in compliance with the relevant laws and
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26 institutional guidelines. The HSP (printed by 16/22 nozzles) and SSP (printed by 16
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29 nozzle) cylinder scaffolds with the size of 8 10 mm were implanted into femoral
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31 bone defects of adult New Zealand rabbits (nine rabbits with defects created both for
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34 right and left posterior limbs, Experimental Animal Center of Shanghai No. 1 Medical
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36 University, Shanghai, China) by surgery. General anesthesia was induced with an
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39 intravenous injection of 20% urethane (4 mL kg1). Critical size defects (8 mm
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diameter, 10 mm length) were transversally created in the interior of the distal femoral
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44 condyle of posterior limbs by a standardized surgical procedure. The defects were
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prepared with an 8.0 mm drill. The depth of the defects was 10 mm as measured by a
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49 digital caliper. Then, one HSP and one SSP scaffold were implanted into the defect of
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right and left posterior limbs in one rabbit, respectively. Afterwards, four rabbits were
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54 sacrificed at 4 or 12 weeks randomly. The femoral-scaffold complex samples (n = 4)
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were collected for Micro-CT analysis immediately. The images were obtained via a
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4 Siemens Inveon Micro PET/CT scanner (Siemens Medical Solution, Germany).
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6 Mineralized tissue was distinguished from non-mineralized tissue via a global
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9 thresholding procedure with a value approximating 1.20 g cm3 (25% lower than 1.6 g
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11 cm3). Only the region in the cylinder of the middle bone defect was measured. Bone
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14 volume in each defect was recorded as the measurement of new bone regeneration.
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16 For histological analysis, all samples were fixed with 4% paraformaldehyde for 2
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19 weeks, and decalcified in 10% EDTA for 3 weeks. Then samples were embedded in
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21 paraffin, and cut using a microtome to yield 300 m thick sections. Afterwards, the
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24 sections were stained with Van Gieson (VG) staining, and then evaluated under light
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26 microscopy (Nikon, ECLIPSE E600).
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29 2.6 Statistic analysis
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31 All data are expressed as means standard deviations (SD) and were analyzed using
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34 one-way ANOVA with p < 0.05 was considered statistically significant.
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39 3. Results and discussion
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3.1 Fabrication and characterization of HSP bioceramic scaffolds
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44 For fabricating these HSP bioceramic scaffolds, two key factors should be carefully
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considered. One is to prepare the printable bioceramic paste (bioprinting ink) and the
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49 other is to design the co-axis (core-shell) printing nozzle. It is known that injectable
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polymer-based bioprinting ink is much easier to prepare than inorganic materials, due
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54 to the inherent rheological characteristic of polymers.22 Compared to polymer-based
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scaffolds, bioceramic scaffolds have generally superior bioactivity for bone
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4 regeneration. However, it is quite difficult for preparing HSP bioceramic scaffolds via
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6 extruding-based 3D printing method since the preparation of printable ink of
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9 bioceramics is one of major challenges. Traditionally, the printable ink of ceramics
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11 (pastes) was mainly prepared by mixing ceramic powders with certain concentration
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14 of polymer solution which could improve the viscosity and printability of ceramic ink.
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16 For example, solid struts packed (SSP) bioceramic scaffolds without hollow structures
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19 were fabricated via 3D printing based on the printable mixture ink of ceramic
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21 powders and polyvinyl alcohol (PVA) or Pluronic F-127 solution.22-25 However, the
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24 rheological characteristic and mechanical strength (stability during printing) of the
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26 prepared bioceramic inks by conventional methods cannot be used for printing HSP
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29 bioceramic scaffolds which requires quite critical printing conditions, mainly
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31 including highly rheological and mechanically stable ceramic paste. Therefore, a
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34 modified printable bioceramic ink was developed in this study by introducing 3-5
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36 wt.% of alginate in this system to improve their rheological and mechanical properties.
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39 Alginate is a biocompatible material, which can form stable hydrogel when contacting
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with dications like Ca2+.20,26 After addition of certain amount of alginate (3-5 wt.%) in
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44 the inks of bioceramic powders and polymer solutions (e.g. 20 wt.% Pluronic
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F-127), a stable bioceramic ink composed of powders, alginate and F-127 was formed.
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49 The mass ratio of ceramic powders, alginate and F-127 plays a critical role for
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preparing the printable bioceramic inks. As shown in Figure 1, after the bioceramic
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54 ink-based scaffolds were printed by 3D co-axis printing method, they were then
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sintered at 1400C for 3 h to remove the alginate and F-127 phases and the
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4 bioceramic particles were densified to form HSP bioceramc scaffolds (Figure 1). The
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6 HSP scaffolds still maintained the designed pore structures including macro pores
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9 (outside of struts) and opened hollow channels (inside of struts) despite that 25% of
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11 longitudinal shrinkage and around 57.8% volume shrinkage happened for the whole
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14 scaffolds after high-temperature sintering at 1400C. XRD analysis indicated that
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16 alginate and Pluronic F-127, as the solution of bioceramic ink, have no obvious
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19 effect on the final crystal phase composition of HSP bioceramic scaffolds which still
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21 maintained pure ceramic phase of Ca7P2Si2O16 (JCPD 11-0676) (Figure 2). According
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24 to JCPD 11-0676, XRD pattern indicated the clear crystal phase of Ca7Si2P2O16. After
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26 sintering under high temperature, the bioceramic has no change of crystal composition.
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29 Besides the preparation of printable bioceramic ink, the design of co-axis nozzle is the
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31 other key factor for printing HSP bioceramics scaffolds. In this study, the co-axis
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34 nozzle was constructed by inserting a right angle stainless steel nozzle with smaller
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36 diameter (core nozzle) into a conic plastic nozzle with larger diameter (shell nozzle)
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39 (as shown in Figure 1). The size and geometries of the hollow struts can be controlled
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by the co-axis nozzle. By using different size of co-axis nozzle, the size of hollow
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44 struts including the outer size, inner size (hollow channel) and the thickness of struts
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could be well controlled (Figure 3). In addition, by designing the shape of inner
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49 nozzle, the geometries of hollow struts could be well prepared with cycle, square and
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even other complicated geometries (Figure 3). By using this methods, other HSP
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54 bioceramic scaffolds such as -tricalcium phosphate, can be effectively prepared. In
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addition, other HSP ceramic scaffolds can be also prepared by using this method,
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4 indicating that the strategy for the modified ceramic ink and the design of co-axis
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6 nozzle stands is an effective method to construct 3D-Printed HSP ceramic scaffolds
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9 not only for biomedical application, but for mechanical engineering and catalysis
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11 application.
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14 The printed HSP bioceramic scaffolds had designed macro pores (outside of struts,
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16 controlled by CAD design) and multi-oriented hollow channel structures (inside of
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19 struts, controlled by core/shell nozzle). The macro pores and hollow channels were
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21 completely opened (Figure 3). Compared to the printed SSP scaffolds, the total
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24 porosity and specific surface area of HSP scaffolds was significantly enhanced. The
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26 HSP scaffolds printed by co-axis nozzle with standard of 20G/27G (shell/core nozzle
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29 standard) had high porosity up to 86% and high specific surface area up to 6500
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31 mm2/g, while the printed SSP scaffolds with similar macro pores by nozzle with
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34 standard of 20G had only 57% of porosity and 2800 mm2/g of specific surface area.
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36 The porosity of HSP scaffolds was nearly 30% higher than that of SSP scaffolds, and
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39 the specific surface area of HSP scaffolds was 2.3 times of that of SSP scaffolds. In
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addition, the porosity of HSP scaffolds can be well controlled from 65 to 85% by
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44 adjusting the core/shell size of the printing nozzles (Figure 4). Therefore, the
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mechanical properties of HSP scaffolds can be effectively controlled by controlling
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49 their porosity via core/shell size ratio, in which the compressive strength (~5MPa) and
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modulus (~160MPa) of HSP scaffolds with core/shell size of 16/23 is comparable
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54 with those of SSP scaffolds (Figure 5). In addition, the compressive strength and
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modulus of HSP scaffolds are 4-6 times of those of bioceramic scaffolds (with similar
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4 porosity) prepared by foam template method,27,28 which is a widely used conventional
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6 method to prepare bioceramic scaffolds for bone tissue engineering. Furthermore, the
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9 compressive strength of HSP scaffolds are comparable with those of 3D printed
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11 hollow channels brushite and monetite scaffolds with lower porosity (only 38% and
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14 44%, respectively) (compressive strength: 7.470.7 and 1.470.2 MPa,
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16 respectively).29 The results suggest that HSP bioceramics scaffolds have significantly
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19 superior mechanical strength as compared to hollow polymer scaffolds prepared by
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21 previously used wire array template method. It is known that the compressive strength
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24 of cancellous bone is about 2-12MPa. The compressive strength of HSP scaffolds is
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26 comparable with that of cancellous bone, suggesting that the strength of HSP
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29 scaffolds is sufficient for non-load bearing application of bone regeneration.
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31 3.2 Attachment and proliferation of cells on printed scaffolds
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34 Our previous studies have confirmed that the prepared bioceramics (Ca7Si2P2O16)
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36 powder, disk and 3D scaffolds had no obvious cytotoxicity and presented good
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39 cytocompatible with bone marrow stromal cells. 19, 21, 24, 30, 31 However, compared to
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those scaffolds without hollow channels structures, one of significant advantages for
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44 the prepared HSP bioceramic scaffolds is that their high porosity and surface area lead
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to distinctively improved cell attachment and migration of bone marrow stem cells
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49 (BMSCs) in the inner of scaffolds. Tissue engineering scaffolds with high porosity
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and surface area were preferred for cell adherence, migration, and exchange of gas
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54 and nutrition as well as tissue regeneration.14,32 In this study, BMSCs were seeded
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through the vertical sectional of HSP scaffolds with different strut size, respectively.
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4 SSP scaffolds were used for the controls. It was found that BMSCs adhered not only
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6 on the outer surface of hollow struts, but also on the inner surface of hollow struts
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9 (Figure 6). Compared to SSP scaffolds with same size of macropores, HSP scaffolds
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11 had significant improved cell attachment at day 3 and proliferation at day 7 (Figure 6).
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14 It is reasonable to speculate that the improved porosity and surface area of HSP
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16 scaffolds mainly contributed to the distinctively high cell proliferation level. In
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19 addition, the hollow channels in the HSP scaffolds may benefit for enhancing oxygen
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21 and nutrients distribution in the inner of scaffolds and further improve cell infiltration
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24 and proliferation in the scaffolds.15,16,33
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26 3.3 Bone formation in vivo
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29 One of most interesting results is that the printed HSP bioceramic scaffolds have
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31 superior bone-forming ability in vivo. According to the obtained results in vitro, the
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34 HSP scaffolds of 16/22 had comparative higher porosity and compressive strength
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36 and modulus than other types of HSP scaffolds. Therefore, In this study, the printed
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39 HSP (nozzle standard: 16/22) bioceramics scaffolds with size of 810 mm (Figure
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7a) were chosen to implant in the femur defect of rabbits (Figure 7b) for investigating
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44 the new bone formation ability in vivo. SSP (nozzle standard: 16) scaffolds with same
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macropores were used for the controls. After implanted for 4 weeks, micro-CT
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49 analysis showed that the HSP scaffolds integrated well with the host bone tissue, and
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new bone tissue started to grow into the porous scaffold, while there was limited new
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54 bone tissue growing into the porous SSP scaffold (Figure 7). Van Gieson (VG)
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staining results indicated that the new bone tissues not only formed in the macropores,
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4 but also grew into the hollow channels of HSP scaffolds (Figure 8). Furthermore, it
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6 was found that there were great amount of newly formed bone tissue formed in the
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9 center of HSP bioceramic scaffolds, while nearly no obviously new bone tissue was
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11 observed in the center of SSP scaffolds at week 4. After 12 weeks of implantation, the
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14 new bone tissue in the hollow channels of HSP scaffold grew much thicker than that
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16 at week 4, and in the meanwhile, the hollow strut wall became much thinner due to
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19 the degradation of scaffolds (Figure 8). Although amount of ions were released and
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21 pore wall was thinned during degradation, the HSP scaffolds still maintained their
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24 regular hollow channel structures to support newly bone tissue ingrowth. Micro-CT
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26 analysis further showed that the bone mineral density (BMD) and bone surface
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29 density of HSP scaffolds were significantly higher than those of SSP scaffolds and
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31 control samples (without scaffolds). All the obtained data showed that the new bone
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34 tissue not only formed in the macropores of HSP scaffolds, but also grew along their
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36 hollow channels of struts, indicating that the hollow channels in the scaffold struts
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39 play an important role to guide new bone formation in vivo.
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Compared to SSP bioceramic scaffolds, which are the general types of scaffolds
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44 obtained via 3D printing, novel HSP bioceramic scaffolds prepared in this work have
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several distinct properties due to their hollow channels structures. First of all, HSP
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49 scaffolds had higher porosity and specific surface area, which provides more space
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and surface for cell infiltration and new bone formation. Secondly, HSP bioceramic
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54 scaffolds had faster degradation rate due to their larger surface area and higher
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porosity. ICP analysis indicated that more ions (Ca and Si) were released from HSP
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4 scaffolds than from SSP scaffolds (Figure 9). Previous studies showed that Si ions had
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6 the ability to promote osteogenesis as compared to -tricalcium phosphate
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9 (-TCP).34-36 In this study, the improved bone regeneration of HSP bioceramic
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11 scaffolds may be directly related to the quick release of Si ions from scaffolds. In
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14 addition, the improved degradation of HSP scaffolds may offer much more room for
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16 growth of new bone tissue.7 Furthermore, the multi-oriented hollow struts with tube
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19 structures provided bridge role for transferring oxygen and nutrition and cells
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21 migration. Compared to mono-oriented hollow channels embed scaffolds created via
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24 template, multi-oriented HSP scaffolds printed in this work could recruit cells from all
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26 around of host tissues and deliver oxygen and nutrition from different directions of
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29 scaffolds, which may benefit the improvement of bone formation.
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31 4. Conclusion
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33
34 In summary, in this work, we presented a useful and facile method for preparing HSP
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36 bioceramics scaffolds with designed macropores and multi-oriented hollow channels
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39 via a modified coaxial 3D printing strategy. The prepared HSP scaffolds combined
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high porosity and surface area with impressive mechanical strength. The unique
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44 hollow-struts structures of bioceramic scaffolds significantly improved cell
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attachment and proliferation, and further promoted formation of new bone in the
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49 center of scaffolds, indicating that HSP ceramic scaffolds can be used for reservation
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of large bone defects. In addition, the strategy can be used for prepare other HSP
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54 ceramic scaffolds (e. g. ZrO2), indicating potential application for tissue engineering,
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mechanical engineering, catalysis and environment materials.
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4 Acknowledgements
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6 This study was funded by the Shanghai Municipal Natural Science Foundation
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8
9 (14ZR1445500), the Natural Science Foundation of China (Grants 31400807,
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11 31370963). The National High Technology Research and Development Program of
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14 China (863 Program, SS2015AA020302) and shanghai outstanding leaders project
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16 (15XD1503900) partly support.
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18
19 References
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9 Bioceramics
10 Coaxis
11 F-127 printing Sintering
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19 Figure 1. The procedure of fabrication of hollow struts-packed (HSP) bioceramics scaffolds:
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21 by mixing bioceramic powders with F-127 solution (concentration: 20 wt.%) and alginate to
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23 prepare printable inks, then HSP bioceramic scaffolds were fabricated via coaxis printing the
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25 prepared inks; the used co-axis printing nozzle was shown in an image (20/27); after sintering,
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27 the bioceramic scaffolds were mechanically stable and the morphologies were designed.
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23 Figure 2. XRD analysis of bioceramic powders and the prepared HSP scaffolds after
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31 Figure 3. SEM images of printed HSP bioceramics scaffolds via core/shell nozzle of 16/22 (a,
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33 e), 18/23 (b, f) and 20/27 (c, g) and printed solid struts-packed (SSP) bioceramics scaffold (d,
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35 h) as control. The strong binding between hollow struts in different layers after sintering (i).
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37 Hollow strut with square (j) morphology, and high magnification of SEM for the dense strut
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39 surface (k). The relationship of struts size for the printed HSP scaffolds with different
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core/shell nozzles of printers (l). The scale bar was 1 mm (a-d), 200 m (e-j) and 20 m (k),
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Figure 4. The porosity (a) and specific surface area (b) of the printed HSP (16/21, 16/22,
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25 16/23, 18/25 and 20/27) scaffolds can be easily controlled by varying the core/shell ratio of
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Figure 5. Compressive strength (a) and Youngs modulus (b) of printed HSP scaffolds with
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44 Figure 6. SEM images of BMSCs seeded in printed HSP bioceramic scaffolds on day
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1 (a, b) and day 7 (c, d). Cells attached on the outside of strut surface of scaffolds (a,
47 c) and in the inner of hollow struts (b, d). Inset images showed the confocal laser
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49 scanning microscope (CLSM) images of cells in scaffolds (blue=DAPI staining). The
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51 proliferation (e) of cells in printed scaffolds. (D16, D18, D20 means solid strut packed
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53 scaffolds with printing nozzle size of 16G, 18G and 20 G, respectively; H16, H18,
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55 H20 means hollow strut packed scaffolds with printing nozzle size of 16/22G, 18/25G
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3 and 20/27G, respectively). White arrows indicate the cells. Scale bar = 50 m. (* p <
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34 Figure 7. Image of printed scaffolds (Left: HSP scaffolds; Right: SSP scaffolds(810 mm)
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36 for in vivo experiment (a); HSP scaffold (hollow) after implanted in the femur of rabbits (b);
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38 Micro-CT analysis of in vivo bone formation ability of HSP scaffolds (c, e) and SSP scaffolds
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40 (d, f) for 4 (c, d) and 12 (e, f)weeks. The BMD (g) and bone surface density (h) of samples
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42 (control: no scaffolds) after implanted in vivo for 12 weeks. It is obvious that hollow
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44 struts-packed bioceramic scaffolds significantly stimulates new bone formation as compared
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to solid struts-packed bioceramic scaffolds (* p < 0.05).
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31 Figure 8. Histological images for SSP (a, d) and HSP (b, c, e and f) scaffolds were taken at
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30 Figure 9. Calcium (a) and silicon (b) ions release from HSP and SSP scaffolds and
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32 weight loss (c) of printed HSP (Hollow) and SSP (Non-hollow) bioceramics scaffolds
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1
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3 The table of contents
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6 Hollow struts packed bioceramics scaffolds with designed macro pores and hollow
7 channels structures are directly fabricated via coaxis printing based on developed inks
8 for guiding bone formation. The hollow scaffolds have increased porosity and surface
9 area for cells adherence, and improved bone formation ability in vivo.
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12 Bioceramics In vitro
13 Ink F-127
14 Alginate
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21 Hollow
22 bioceramics
23 scaffold
24 Coaxis printing
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