Calcium silicate ceramic scaffolds toughened with hydroxyapatite whiskers for bone tissue engineering

    Calcium silicate ceramic scaffolds toughened with hydroxyapatite whiskers for bone tissue engineering Pei Feng, Pingpin Wei, Pengjian Li, Chengde Gao, Cijun Shuai, Shuping Peng PII: DOI: Reference:

S1044-5803(14)00258-7 doi: 10.1016/j.matchar.2014.08.017 MTL 7669

To appear in:

Materials Characterization

Received date: Revised date: Accepted date:

23 December 2013 18 August 2014 20 August 2014

Please cite this article as: Feng Pei, Wei Pingpin, Li Pengjian, Gao Chengde, Shuai Cijun, Peng Shuping, Calcium silicate ceramic scaffolds toughened with hydroxyapatite whiskers for bone tissue engineering, Materials Characterization (2014), doi: 10.1016/j.matchar.2014.08.017

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ACCEPTED MANUSCRIPT Calcium silicate ceramic scaffolds toughened with hydroxyapatite whiskers for bone tissue engineering Pei Fenga,＃, Pingpin Weic,＃, Pengjian Lia, Chengde Gaoa, Cijun Shuaia, b,*, Shuping Pengc,*

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State Key Laboratory of High Performance Complex Manufacturing, Central South University, Changsha, P. R. China, 410083

Department of Regenerative Medicine & Cell Biology, Medical University of South Carolina,

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b

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a

Charleston, SC, 29425

Cancer Research Institute, Central South University, Changsha, P. R. China, 410078

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c

* Corresponding author: Cijun Shuai, Tel: (86)-731-88879351, Fax: (86)-731-88879044,

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e-mail: [email protected]; Shuping Peng, Tel: (86)-731-84805412, Fax: (86)-731-88879044, e-mail: [email protected] ＃

These authors contributed equally to this work.

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Abstract:

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Calcium silicate possessed excellent biocompatibility, bioactivity and degradability, while the high

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brittleness limited its application in load-bearing sites. Hydroxyapatite whiskers ranging from 0 to 30 wt.% were incorporated into the calcium silicate matrix to improve the strength and fracture resistance. Porous scaffolds were fabricated by selective laser sintering. The effects of hydroxyapatite

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whiskers on the mechanical properties and toughening mechanisms were investigated. The results showed the scaffolds had a uniform and continuous inner network with the pore size ranging between 0.5 mm and 0.8 mm. The mechanical properties were enhanced with increasing hydroxyapatite whiskers, reached a maximum at 20 wt.% (compressive strength: 27.28 MPa, compressive Young’s modulus: 156.2 MPa, flexural strength: 15.64 MPa and fracture toughness: 1.43 MPa·m1/2) and then decreased by more addition of hydroxyapatite whiskers. The improvement of mechanical properties was due to whiskers pull-out, crack deflection and crack bridging. Moreover, the degradation rate decreased with the increase of hydroxyapatite whiskers content. A layer of bone-like apatite was formed on the scaffolds surfaces after soaked in simulated body fluid. Human osteoblast-like MG-63 cells spread well on the scaffolds and proliferated with increasing the culture time. These findings suggested that the calcium silicate scaffolds reinforced with hydroxyapatite whiskers showed great potential for bone regeneration and tissue engineering applications.

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ACCEPTED MANUSCRIPT Key words: Calcium silicate; Hydroxyapatite whiskers; Porous scaffolds; Mechanical properties 1. Introduction Calcium silicate (CaSiO3, CS) was widely used for bone substitutes in clinical applications due to

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the significant biocompatibility, high osteoconductivity and excellent angiogenesis-induced ability

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[1,2]. Nevertheless, the foremost drawback of CS ceramic was lack of sufficient mechanical strength

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and resistance, which impeded the application in load-bearing bone [3]. Some methods were developed to toughen CS ceramic, such as by enhancing densification, adding dopants and controlling

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microstructure [4-6]. Whisker or fibrous materials were found to be effective in strengthening and toughening ceramic materials through crack deflection, crack bridging, pinning and whisker pull-out,

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which could absorb the energy of crack propagation and eliminate stress concentration at the crack tip [7-9]. Some whiskers such as SiC, Si3N4, Al2O3, ZrO2 and carbon had been applied to enhance the strength and toughness of ceramics [10-13]. Unfortunately, those bioinert reinforcements improved

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the mechanical properties at the sacrifice of the biocompatibility and bioactivity of bioceramic.

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Hydroxyapatite (Ca10(PO4)6(OH)2, HA) whiskers possessed chemical composition, crystal structure

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and Ca:P ratio (1.67) similar to apatite of human bones. The whiskers had shown good bonding ability to bone through the development of a rich HA layer on their surface. In addition, the whisker-like morphology was helpful in improving mechanical properties [14-16]. Kane RJ [17] investigated the

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effect of HA reinforcement on mechanical properties of freeze-dried collagen scaffolds and found that 61 vol% HA whiskers resulted in up to a ten-fold increase in compressive modulus. Converse GL [18] studied

polyetheretherketone

(PEEK)

reinforced

with

0-50

vol.%

HA

whiskers

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compression-molded and found that the whiskers effectively increased the elastic modulus and tensile strength. Suchanek W [19] reported 0~30 wt.% HA whiskers reinforced HA composite by hot isostatic pressing (HIP). The HA whiskers reinforced the composites and exhibited significantly improved toughness without decrease of bioactivity and biocompatibility. A recent study by Hu H showed HA whiskers simultaneously increased the compressive strength of β-TCP ceramics and improved the cell adhesion and proliferation [20]. Therefore, HA whiskers could act not only as reinforcement, but also as a bioactive phase in bone substitute materials. This strategy had proved to be quite successful for polymer or dense ceramic, while, to our best knowledge, a few research works had been carried out on HA whiskers reinforced porous ceramic scaffold [20,21].

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ACCEPTED MANUSCRIPT It was well known that a high degree of porosity and an appropriate pore size of a scaffold were necessary to provide adequate space for tissue formation as well as for the diffusion of nutrients, gases and growth factors [22,23]. Nowadays, a number of fabrication techniques had been developed

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to manufacture porous ceramic scaffold, such as selective laser sintering (SLS) [24], solid freeform

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fabrication (SFF) [25] and fused deposition modeling (FDM) [26]. Among them, SLS was particularly

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attractive because of its ability to fabricate scaffold with not only customized and complex external shapes but also internal pore architectures by controlled layer-by-layer plotting. In addition, the rapid

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heating and cooling process of SLS can reduce the decomposition of HA due to shorter exposure at high temperature.

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Up to now, few works have been carried out to fabricate the porous CS scaffolds using SLS and to enhance the mechanical properties by addition of HA whiskers at the same time. Our previous study has fabricated the porous calcium phosphate ceramic scaffolds with different weight ratios of

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TCP/HAP (0/100, 10/90, 30/70, 50/50, 70/30 and 100/0) via SLS [27]. The degradation rate and

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mechanical properties of the scaffolds were investigated. In the paper, HA whiskers were used as

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reinforcement to CS scaffold for bone tissue engineering. Porous scaffolds were prepared using SLS technology. The phase compositions and microstructure of the scaffolds were investigated. The effect of HA whiskers on the fracture toughness and compressive strength was assessed. The degradation

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behavior and apatite-forming ability of the scaffolds was performed in phosphate buffered saline (PBS) and simulated body fluid (SBF), respectively. In addition, MG-63 cells attachment and proliferation on the scaffolds were also evaluated. 2. Materials and methods 2.1. Materials and scaffolds The CS powder with diameter of 0.1 to 2 μm was purchased from Kunshan Huaqiao New Materials Co., Ltd. (China). The CS had a total metal content (mg/kg) ≤50, thereinto As≤3, Cd≤5, Hg≤5, Pb≤30. The HA whiskers with approximately 500 nm in length, 10 nm in width and an aspect ratio of 50 were purchased from Nanjing Emperor Nano Material Co. China. CS powder was mixed with HA whiskers in various proportions (0, 5, 10, 20, 30 wt.%) by mechanical mixing. In detail, the compounds were firstly milled in an agate mortar for 30 min, and then appropriate amounts of ethanol were added to the mixtures followed by ball milling for 8 h at room temperature. After milling, the compounds were

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ACCEPTED MANUSCRIPT dried at 80 °C for 24 h in an electrothermal blowing dry box (101-00S, Guangzhou Dayang Electronic Machinery Equipment Co., Ltd, China). The porous scaffolds were fabricated on a homemade selective laser sintering system with optimal

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SLS process parameters: laser power of 10 W, spot diameter of 1 mm, sintering speed of 100 mm/min,

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hatch distance of 2.5 mm and layer thickness of 0.1 mm. Core features of the SLS system included the

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optical focusing system, the sintering system, the control system and the three-dimensional motion platform. The optical devices (Haas Co., USA) were used in the optical focusing system to focus the

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incident optical beam into a spot with a small size. The minimum spot diameter could reach 50 µm. The CO2 laser (model Firestar® t-Series, Synrad Co., USA) of the sintering system had a maximum

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power output of 100 W. The three-dimensional motion platform and the laser sintering parameters were controlled by a 6050 motion control card (Dong Fang Jia Hong Co.). After sintering, the excess powder in the exterior and internal architecture was brushed off.

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2.2. Microstructure

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The phase composition was determined by X-ray diffraction analysis (XRD) (D8-ADVANCE

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Bruker AXS Inc., Germany) with Cu-Kα radiation. The accelerating voltage and current were 40 kV and 40 mA, respectively. Samples were grinded into powders and tested with 2θ values from 20° to 60° at a step size of 0.02°. Phases were identified by comparing the diffraction patterns with the

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standard PDF cards.

The fracture morphology of the scaffolds was observed by scanning electronic microscopy (SEM, FEI Quanta-200, USA). The fracture surface of scaffolds were fixed on a copper stud and coated with gold for SEM observation to investigate the morphologies and whiskers distributions. 2.3. Mechanical characterization The apparent density of the scaffold samples (15 ×15 ×7 mm3) was measured with Archimedes method. The relative density was defined as the percentage of the apparent density to their corresponding theoretical density of the composite powders. The theoretical densities of CS and HA powders were 2.91 g/cm3 and 3.16 g/cm3, respectively. The compressive strength of the different scaffolds (15 ×15 ×7 mm3) were measured using a universal testing machine (WD-D1, Shanghai Zhuoji instruments Co. LTD, China) at a crosshead speed of 0.5 mm/min. During the compressive test, the load and displacement were monitored and recorded. The Young’s modulus (E) of the scaffolds

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ACCEPTED MANUSCRIPT was calculated from the compressive test curves. The flexural strength of the scaffold samples (25 ×15 ×5 mm3) was measured at room temperature by a three point bending method on a universal testing machine (WD-D1, Shanghai Zhuoji instruments Co. LTD, China) with a fixture span of 20

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mm and a crosshead speed of 0.5 mm/min.

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The fracture toughness (KIC) was evaluated by an indentation method using a Vickers hardness

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tester (HXD-1000TM/LCD, Digital Micro Hardness Tester, Shanghai Taiming Optical Instrument Co. Ltd). Each scaffold was ground and polished using diamond paste before the test. A load of 300 gf

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was applied in each scaffold for 15 s. Fracture toughness KIC was derived from the model proposed by Evans and Charles [28], as shown in equation (1). The average value for each test was taken for five

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samples to verify the results.

KIC = 0.0824Pc-3/2

(1)

where KIC is the fracture toughness (MPa·m1/2), P is the applied load (N), and c is the diagonal crack

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length (m) (measured from the center of the indent).

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2.4. Apatite-forming ability in SBF

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The apatite-forming ability of the scaffolds (15 ×15 ×7 mm3) was evaluated by examining the bone-like apatite formation on their surface in SBF. The SBF solution was prepared by dissolving reagent-grade chemicals of NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, CaCl2 and Na2SO4 in

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distilled water and buffered at pH 7.4 with tris-hydroxymethyl-aminomethane (Tris) and HCl. The SBF was prepared according to the procedure described by Kokubo [29]. The solution had similar ion concentrations to those of human blood plasma, as shown in the Table 1. The experimental was carried out according to the procedure described by Lei [30]. The scaffold samples were soaked in the SBF for 7 days at 37 ℃. The ratio of the solution volume to the each sample mass was 200 ml/g. The SBF solutions were refreshed every 3 days. After immersed for designed days, the scaffolds were taken out, gently washed with distilled water and then dried at room temperature. The morphology of apatite formed on the scaffolds surface was investigated by SEM. The elemental composition and Ca/P ratio of the apatite were determined by EDX. 2.5. Degradation in PBS In vitro degradation of scaffold samples (15 ×15 ×7 mm3) was carried out in PBS solution under pH 7.4 at 37 ℃ for 7 days. Five prewetted scaffolds of each type were placed in a glass bottle filled

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ACCEPTED MANUSCRIPT with PBS. The ratio of solution volume to scaffolds mass was 200 ml/g. The solution was renewed every 3 days. After soaking, the samples were taken out, gently washed with distilled water and then dried at room temperature for 1 day. The changes in the microstructure of the samples surface were

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observed with SEM.

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2.6. Cell culture

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MG-63 cells were obtained form American Type Culture Collection (ATCC, Rockville, MD) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA). The medium was

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supplemented with 10% fetal bovine serum (FBS), 5% penicillin/streptomycin, 5% glutamine, incubated at 37 °C and 5% CO2, and maintained according to the cell line specific recommendation of

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the ATCC. Cells were cultured in a humidified 37 ◦C/5% CO2 incubator and the culture medium was changed every day. Passage 3 MG-63 cells were collected by treatment with trypsin/EDTA (Gibco, USA) solution for cell culture. The scaffolds (15 ×15 ×7 mm3) were immersed in the cell culture

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medium for 24 h before being used for the subsequent cell seeding assays. A seeding density of 2×10 4

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cells/cm2 was applied for attachment and proliferation assays on the scaffolds. The scaffolds after

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cultured with MG-63 cells for 1, 3, 5and 7 days were fixed in 2.5% glutaraldehyde overnight at 4 °C. After being washed twice with PBS solution, the scaffolds were dehydrated in a graded series ethanol for 10 min and critically point dried. After that, samples were sputter-coated with gold and imaged

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with SEM (Tescan, Mira 3 FEG-SEM, TESCAN Co., Czech) to observe the adhesion and proliferation of MG-63 cells on the scaffolds. 2.7. Statistical analysis

The data were expressed as means ± standard deviation (SD) for all experiments and were analyzed using One-Way ANOVA with a Post Hoc test. A p-value <0.01 was considered statistically significant. 3. Results and discussion 3.1. Porous scaffold The SEM and transmission electron microscopy (TEM) images of the starting powder and composite powder were shown in Figs.1 (a-c). The HA whiskers were approximately 500 nm in length and 10 nm in width from the TEM image. They were uniformly adhered to CS particles in the composite powder. The scaffold had a tetragonal geometry with size of 13×13×10 mm3 (length×width×height). The isometric view, top view and front view of the scaffolds were presented in

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ACCEPTED MANUSCRIPT Figs.1 (d-f). The pores were fully interconnected and distributed throughout the whole scaffold with size of about 0.5~0.8 mm. Besides, good connections between pore walls were observed. 3.2. Microstructure

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The XRD patterns of the starting powders and sintered specimens were presented in Fig. 2. The

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starting CaSiO3 powders were identified as β-CaSiO3 (PDF Card no. 84-0654) without any other

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phase (Fig.2 a). The diffraction patterns of the starting HA whiskers were in good agreement with standard hydroxyapatite PDF Card (no. 09-432) (Fig.2 b). The XRD pattern of CaSiO3 after sintering was presented in Fig. 2 (c). It was observed that a majority of β-CaSiO3 was transformed to α-CaSiO3

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(PDF Card no.74-0874). It had been reported that β-CaSiO3 transformed into α-CaSiO3 at high

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temperature [31]. The pattern of HA/CS composite powders after sintering (Figs.2 d-g) were composed of HA, β-CaSiO3 and α-CaSiO3, which demonstrated that there was no reaction occurred between HA whiskers and CaSiO3. What’s more, it could be acknowledged that HA whiskers were

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preserved during sintering. Three diffraction peaks at Bragg angles 2θ=25.8°, 28.8° and 32.8° could

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be observed, which were attributed to the diffraction of the (002) lattice plane, the (210) lattice plane

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and the (300) lattice plane of HA, respectively [32,33]. The fracture surfaces of the scaffolds were shown in Fig.3. The scaffold with 0 wt.% HA whiskers showed a smooth and dense surface (Fig.3 a). It could be seen that the HA whiskers were dispersed in

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CS ceramic matrix with original appearance after sintering (as marked with red arrows in Fig.3 b). There were some HA whiskers pull-out and HA whiskers were well bonded with the CS matrix. The whiskers pull-out became more obvious with increasing HA whiskers content (Figs.3 c,d). Meanwhile, it was observed that the grain boundaries became rough, indicating HA whiskers resulted in a transgranular fracture mode. For the scaffolds with 10 wt.% and 20 wt.% HA whiskers, the grains fracture showed obvious transgranular fracture (as circled in Figs.3 (c,d)). While 30 wt.% HA whiskers were added, the transgranular fracture was disappeared. Simultaneously, large agglomerates appeared and a loose structure with some cavities was formed (Fig.3 e). If the scaffold withstands the load, some stripping would happen. As a result, the strength would drop down. The indentation induced crack propagation of a polished surface was shown in Fig.4 (a). Crack bridging, crack deflection and pull-out were observed in radial cracks stemming from micro hardness indents. With regard to whiskers bridging (Fig.4 b), the HA whiskers consumed fracture energy by

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ACCEPTED MANUSCRIPT spanning wakes of the cracks, and consequently improved fracture toughness of the scaffolds. As shown in Fig.4 (c), a crack propagated and deflected around the HA whiskers along the interface. The crack deflection also played an important role in absorbing crack propagating energy during fracture.

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The main toughening mechanisms of whiskers were crack bridging, crack deflection and whiskers

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pull-out. These toughening effects could be related to the energy-dissipating processes at the crack tip

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[34]. When the crack size was small, the crack bridging played a chief role. The microcracks were bridged by whiskers at fracture tip area. If a closure stress was loaded on the crack area, the

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continuous propagates of microcracks would be inhibited. Then cracks couldn’t continue to grow until the whiskers were broken. HA whiskers were observed to bridge the crack on the fracture area, as

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shown in Fig.4 (b). The crack was crossed and blocked by the HA whiskers. If cracks came across whiskers with a small angle, they would deflect and extend along the interface between whiskers and matrix, instead of propagating in the original direction. As a result, the cracks growth paths were

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lengthened and more energy would be consumed in the process of crack growth.

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As the stress and size of the displacement increased, the whiskers pull-out became the primary

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toughening mechanism. If the crack surfaces met whiskers orientation with a large angle, the whiskers would be pulled out under the shearing stress, which was higher than the shear yield strength of ceramic matrix. As shown in Fig.3, HA whiskers perpendicular to the fracture surface were pulled out

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of the ceramic matrix. It was believed that the toughness of ceramic could be effectively improved due to additional resistance from the drag by whiskers [35]. The combined effect of the three toughening mechanisms optimized the stress transfer and stress distribution in scaffolds. So the strength and toughness of scaffolds were improved. 3.3. Mechanical behavior The densities of the scaffolds with different content of HA whiskers were shown in Table 2. It could be seen that the relative density of scaffold with 0 wt.% HA whiskers was 82.96% and increased to 92.26% with 20 wt.% HA whiskers. The increase of relative density could be related to a more homogeneous distribution of HA whiskers which have much smaller size than CS, and acted as particulate inclusions to fill the pores existing between CS particles [36]. The relative density of scaffold decreased to 91.65% with 30 wt.% HA whiskers. The decrease was due to the agglomeration of HA whiskers [37].

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ACCEPTED MANUSCRIPT An example of compressive curves of five different specimens was shown in Fig.5 (a). It was observed that compression tests presented similar stress-strain behavior for all scaffolds. Their compressive strength increased almost linearly with the deformation. Then the scaffolds exhibited

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brittle failure, which was characterized by a sudden fracture without significant plastic deformation.

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Nevertheless, the compressive strength was enhanced by the addition of HA whiskers. The

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compressive strength and Young’s modulus of all scaffolds were presented in Fig.5 (b). The compressive strength and Young’s modulus increased with HA whiskers to 20 wt.% and then

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decreased with further increase in the HA whiskers. The highest compressive strength and Young’s modulus were obtained for the scaffold with 20 wt.% HA whiskers. The compressive strength and

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Young’s modulus were improved from 18.19±1.25 MPa to 27.28±0.70 MPa and 117.5±17.8 MPa to 156.2±11.9 MPa, respectively. The compressive strength of cancellous bone and cortical bone was 2-12 MPa [38] and 130-180 MPa [39], respectively. Although the compressive strength of scaffold

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was lower than that of cortical bone, it was much higher than that of cancellous bone. Previous studies

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showed that the compressive strength of CS scaffolds was quite low (<1 MPa) [40-42]. A recent study

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has shown that the compressive strength of 3-D plotted CS scaffolds could reach 4 MPa [43]. The variation of mechanical properties was mainly associated with whiskers content and their distribution in ceramic matrix. On the one hand, the improvement of compressive strength and

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Young’s modulus were attributed to aforesaid three toughening effects. On the other hand, a good distribution of HA whiskers and interfacial bonding with the matrix conferred the enhanced mechanical properties. However, there was a negative influence on the mechanical properties with further increase in the HA whiskers content. This was due to a weak interface and an inhomogeneous distribution of whiskers. Variation of the flexural strength with whiskers fraction in the range from 0 to 30 wt% was shown in Fig.6 (a). The flexural strength of the scaffold increased from 2.83 to 15.64 MPa with the increase in HA whiskers content from 0 to 20 wt.%. The flexural strength was reduced to 11.81 MPa when the content was further increased to 30 wt.%. Microindentation tests were carried out to evaluate the fracture toughness of the scaffolds (Fig.6 b). Fracture toughness of the CS scaffold was just 1.19±0.09 MPa·m1/2. The fracture toughness was slightly improved for scaffold with 5 wt.% HA whiskers. The fracture toughness increased to 1.29±0.09 MPa·m1/2 with HA whiskers increasing to 10 wt.%. On

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ACCEPTED MANUSCRIPT further increasing HA whiskers to 20 wt.%, the fracture toughness followed the increasing trend and reached a maximum of 1.43±0.05 MPa·m1/2. However, the fracture toughness decreased when HA whiskers were more than 20 wt.%. This was similar to the situation of compressive strength. The

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improvement of fracture toughness was due to crack deflection, crack bridging and pull-out

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mechanism. While the whiskers were more than 20 wt.%, it was difficult to homogeneously dispersed

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the whiskers in the matrix, as shown in Fig.4(e). The agglomeration of HA whiskers resulted in a loose bonding. Thereby, toughening effects was decreased.

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3.4. Apatite-forming ability in SBF

The scaffolds were soaked in SBF to determine the apatite-forming ability. It was apparent that all

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scaffolds surface were covered with many mineralizations after immersion in SBF for 7 days, as shown in Fig.7. Plenty of rod-shaped crystals (~2 µm in length and ~500 nm in diameter) appeared on the surface of the scaffold with 0 wt.% HA whiskers (Fig.7 a). For scaffold with 5 wt.% HA whiskers,

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the morphology of the agglomerates changed apparently. As shown in Fig.7 (b), some ball-like

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particles were distributed on the surface. A large amount of ball-like particles were aggregated with

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HA whiskers gradually increasing (Figs.7 c-e). And the scaffold with 30 wt.% HA whiskers was totally covered by the newly formed layer. The high-magnification images showed that the deposits were composed of crystals in a typical worm-like morphology. In this study, the CS scaffolds in

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combination with HA whiskers showed excellent apatite mineralization, which indicated that incorporating HA whiskers into CS scaffolds didn’t impair the apatite-forming ability of CS scaffolds. Previous studies have showed that nanosized HA had a very high surface area to volume ratio, which could significantly increase the bioactivity of biomaterials [44-46]. Moreover, the addition of HA whiskers provided more nucleation sites for the apatite crystals as the content of HA increased. XRD pattern of the scaffold with 20 wt.% HA whiskers after soaking in SBF for 7 days was shown in Fig.8 (a). It could be seen that the CS and HA characteristic peaks existed in the scaffold after soaking in SBF. The peak intensity of the CS decreased, while the peak intensity of the HA phase increased as compared with the peak intensity of the scaffold with 20 wt.% HA whiskers before soaking in SBF. EDX spectras of the scaffold with 0 wt.%, 5 wt.% and 30 wt.% HA whiskers after soaking in SBF were shown in Figs.8 (b-d). The compound on the scaffold surface was mainly constituted by Ca, P, Si and O. However, peaks for Na, Cl and C were also detected. The presence of

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ACCEPTED MANUSCRIPT Na and Cl could be explained taking into account that the elements were present in the SBF [47], while the presence of C was due to the CO32- substitute the PO43- in apatite [48]. Change in Ca/P ratio of the scaffolds with different HA whiskers after soaking in SBF for 7 days was shown in Fig.8 (e).

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The Ca/P ratio was 1.23 for the scaffold with 0 wt.% HA whiskers. It increased to 1.72 with increase

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in HA whiskers content to 30 wt.%, which was similar to the standard Ca/P ratio of 1.67 for HA [49].

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The results of XRD and the EDX indicated that the bone-like apatite layer was formed on the scaffold surface. It had been found that bone-like apatite on the scaffolds played an important role in maintaining their bioactivity. It was helpful to bond with the host bone when implanted in the living

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body. What’s more, the scaffolds with bone-like apatite formed on their surface possessed the capacity

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to enhance the osteoblastic activity, including proliferation and differentiation [50]. 3.5. Degradation in PBS

The surface morphologies of the scaffolds before and after immersion in PBS for 7 days were

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shown in Fig.6. There was a smooth and dense surface before immersion in PBS (Fig.9 a). The

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surface morphologies changed significantly after immersion in PBS. The scaffold surface was covered

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by a dense apatite layer when the HA whiskers content was 0 wt.% (Fig.9 b). Dissolution and precipitation behaviors could be seen for the scaffolds with more HA whiskers (Figs.9 c-f). The dissolution ability became poor with the increase of HA content. Previous studies showed that the

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scaffolds made of HA have a slow degradation rate [51,52], while CS, serving as the matrix material in this study, is bioresorbable and has a fairly fast degradation rate than HA [53,54]. The degradation rate of scaffold could be tailored by adding different HA content to CS. It is well known that proper degradation rate was one of the most important characteristic that a scaffold must fulfill. 3.6. Cell morphology and proliferation The morphological features of MG-63 cells cultured on the scaffold with 20 wt.% HA whiskers for different days were shown in Fig.10. Cells with irregular morphology were observed to spread well with an intimate contact with the scaffold surface after 1 day (Fig.10 a). The cells adhered tightly to the scaffold surface and the number was increased after 3 days (Fig.10 b). The cells exhibited a high degree of density after 5 days (Fig.10 c). Furthermore, they partially covered the scaffold surface and neighboring cells maintained physical contact through multiple extensions. The cells covered the whole scaffold surface and the cell-cell interactions with long cytoplasmic extensions were observed

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ACCEPTED MANUSCRIPT after 7 days (Fig.10 d). These results suggested that the scaffold with 20 wt.% HA whiskers had good cytocompatibility to support MG-63 cells adhesion and growing. Previous studies showed that the Si and Ca ions released from bioactive CS promoted osteoblast cell adhesion and proliferation in vitro

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[55,56].

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4. Conclusions

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HA whiskers were incorporated into CS scaffolds in order to improve the mechanical properties. The porous scaffolds were fabricated by selective laser sintering. The scaffolds possessed highly porous

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structure with 0.5~0.8 mm pore size and fully interconnected pore network. Moreover, it was found that HA whiskers were embedded in the ceramic matrix with good bonding ability on the fracture surfaces

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of scaffolds. The compressive strength, compressive Young’s modulus and fracture toughness were enhanced with HA whiskers ranging from 0 to 20 wt.%. Subsequently, it declined due to large agglomerates of HA whiskers. The mechanical properties were increased but lower than those of

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cortical bone. The toughening mechanisms of crack bridging, crack deflection and whiskers pull-out

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were observed. The addition of HA whiskers in CS could slow the degradation rate of scaffolds.

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Meanwhile, the scaffolds exhibited good apatite-forming ability in vitro SBF experiment and excellent support for cell attachment. Considering the results obtained, HA whiskers reinforced CS scaffolds showed great prospect in bone tissue engineering.

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Acknowledgements

This work was supported by the following funds: (1) The Natural Science Foundation of China (51222506, 81372366); (2) Hunan Provincial Natural Science Foundation of China (14JJ1006); (3) Project supported by the Fok Ying-Tong Education Foundation, China (131050); (4) Shenzhen Strategic Emerging Industrial Development Funds (JCYJ20130401160614372); (5) The Open-End Fund for the Valuable and Precision Instruments of Central South University; (6)The faculty research grant of Central South University (2013JSJJ011, 2013JSJJ046); (7) State Key Laboratory of New Ceramic and Fine Processing Tsinghua University (KF201413); (8) Program for New Century Excellent Talents in University (NCET-12-0544); (9) The Fundamental Research Funds for the Central Universities of Central South University.

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Figure captions Fig.1 (a) SEM micrograph of the CS powder, (b) TEM image of the HA whiskers, (c) SEM

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micrograph of the composite containing 10 wt.% HA whiskers and (d-f) stereoscope images of a

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representative scaffold

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Fig.2 XRD patterns of (a) starting HA whiskers, (b) starting CaSiO3, (c) CaSiO3 with 0 wt.% HA whiskers after sintering, (d) HA/CS composite powders (d, 5 wt.% HA, e, 10 wt.% HA, f, 20 wt.%

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HA, g, 30 wt.% HA) after sintering. (HA, JCPDS 09-0432; β-CS, JCPDS 84-0654; α-CS, JCPDS 74-0874)

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Fig.3 SEM images of fractured surfaces of HA whiskers reinforced CS: (a) 0 wt.% HA (b) 5 wt.% HA (c) 10 wt.% HA (d) 20 wt.% HA and (e) 30 wt.% HA

Fig.4 SEM images of (a) a vickers indentation obtained on the surface of scaffold, (b) crack bridging

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Fig.5 (a) The relationship between compressive strength and deformation during mechanical testing

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(A 0 wt.% HA, B 5 wt.% HA, C 10 wt.% HA, D 20 wt.% HA and E 30 wt.% HA), (b) Compressive strength and Young’s modulus as a function of HA whiskers Fig.6 Fracture toughness of the scaffolds with different HA whiskers contents

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Fig.7 SEM images of the surfaces morphology after immersion in SBF for 7 days: (a) 0 wt.% HA, (b) 5 wt.% HA, (c) 10 wt.% HA, (d) 20 wt.% HA and (e, f) 30 wt.% HA (e, magnification 4000× and f, magnification 40000×)

Fig.8 (a) XRD pattern of the scaffold with 20 wt.% HA whisker after soaking in SBF for 7 days, (b-d) EDX analysis of the scaffold with 0 wt.% (b), 10 wt.% (c) and 20 wt.% (d) HA whisker after soaking in SBF, (e) changes in Ca/P ratio of the scaffolds with different HA whisker after soaking in SBF Fig.9 SEM images of the surfaces morphology before (a) and after immersion in PBS for 7 days (b-f): (b) 0 wt.% HA, (c) 5 wt.% HA, (d) 10 wt.% HA, (e) 20 wt.% HA and (f) 30 wt.% HA Fig.10 SEM micrographs of the scaffold with 20 wt.% HA whiskers seeded with MG-63 cells after 1 (a), 3 (b), 5 (c) and 7 days (d) in culture

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Tables

K+

Ca2+

Mg2+

Cl-

142.0

5.0

2.5

1.5

148.8

142.0

5.0

2.5

1.5

103.0

SBF mmol/L

HPO4-

SO42-

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27.0

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Table 1 Ion concentration of SBF solution and human blood plasma (HBP)

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HA whiskers content (wt.%)

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Table 2 Densities of the scaffold with different HA whiskers content 5

10

20

30

2.414

2.466

2.609

2.728

2.762

Relative density (%)

82.96

84.39

88.95

92.26

91.65

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Apparent density (g/cm3)

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Highlights  HA whiskers were incorporated into CS to improve the mechanical properties  HA/CS scaffolds were successfully fabricated by selective laser sintering  The improvement of mechanical properties was due to whiskers pull-out, crack deflection and crack bridging  The CS scaffolds in combination with HA whiskers showed excellent apatite forming ability

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