Toughening and strengthening mechanisms of porous akermanite scaffolds reinforced with nano-titania

Abstract

Akermanite possesses excellent biocompatibility and biodegradability, while low fracture toughness and brittleness have limited its use in load bearing sites of bone tissue. In this work, nano-titania (nano-TiO₂) was dispersed into the ceramic-matrix to enhance the mechanical properties of porous akermanite scaffolds fabricated with selective laser sintering (SLS). The fabrication process, microstructure and mechanical and biological properties were investigated. The results showed that the nano-TiO₂ particles were dispersed both within the akermanite grains and along the grain boundaries. The grain size of akermanite was refined due to the pinning effect of the nano-TiO₂ particles on the grain boundaries. The crack deflection around the nano-TiO₂ particles was observed due to the mismatch of thermal expansion coefficients between TiO₂ and akermanite. The fracture mode changed from intergranular fracture to more and more transgranular fracture as the concentration of nano-TiO₂ increased from 0 to 5 wt%. Meanwhile, the fracture toughness, Vickers hardness, compressive strength and stiffness were significantly increased with increasing nano-TiO₂. The improvement of mechanical properties was due to the grain size refinement, the crack deflection, as well as the fracture mode transition. The bone like apatite was formed on the scaffolds in simulated body fluid (SBF). The human osteoblast-like MG-63 cells (MG-63 cells) adhered and grew well on the scaffolds. The porous akermanite scaffolds reinforced with nano-TiO₂ have considerable potential for application in bone tissue engineering.

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1 Introduction

Bone tissue engineering has a great potential for repairing bone defects.¹ Scaffolds play a vital role in bone tissue engineering as they provide a framework and initial support for cell attachment, proliferation and differentiation. An ideal scaffold should possess good biocompatibility, adequate mechanical properties and controllable degradation rates.² Bioceramics are one of the most potential scaffold materials due to the biocompatibility and biodegradability. Akermanite [Ca₂MgSi₂O₇], as a Ca-, Si-, Mg-containing bioceramic, has been demonstrated to be osteoinductive and able to promote bone repair.^3,4 It can release Ca, Si and Mg ions after implantation in the body. These ions can enhance osteoblasts adhesion, proliferation and differentiation.^5,6 Moreover, akermanite has a controllable degradation rate and can form a bone like apatite layer after immersion in simulated body fluid.^7,8 The preliminary studies indicated that akermanite has great potential as a scaffold material. However, the mechanical properties are much lower than the human cortical bone, which limits the application in heavy loaded implants.^9–11 Therefore, it is necessary to further increase the mechanical properties of akermanite.

The mechanical properties of ceramics are known to be improved significantly by dispersing nanometer sized particles such as metal oxides,^12–14 carbon nanotube,^15–17 bioglass (BG)^17–20 into the ceramic-matrix. Among the many reinforcements used in previous studies, TiO₂ have attracted considerable attention mainly due to the excellent biocompatibility and high mechanical characteristics.^21,22 Khalil et al.²³ used TiO₂ as reinforcement to improve the mechanical properties of hydroxyapatite (HAP) and found that the addition of 5 wt% TiO₂ could improve the fracture toughness and compressive strength of sintered compacts. Kailasanathan et al.²⁴ prepared nano-HAP/gelatin composites with incorporation of TiO₂ ceramics as reinforcing phase and found that the compressive strength of the TiO₂ enhanced scaffolds increased more than twice than nano-HAP/gelatin composites. Seeley et al.²⁵ fabricated the tricalcium phosphate (TCP) compacts with different TiO₂ content and found that the presence of TiO₂ in TCP improved densification and compression strength. Despite these researchers have used TiO₂ to improve the mechanical properties of bioceramics, the details of the mechanisms leading to toughening and strengthening was still unclear.

In addition, Scaffolds should possess an appropriate porous structure (pore size, porosity, pore distribution and geometry shape),^26–28 which is critical to offer good conditions for cells growth and flow transport of nutrients and metabolic waste after the implantation. The technique of SLS can be used to manufacture a 3D porous scaffold with customized external shape.^29,30 Moreover, it can realize the accurate control of porous structure to meet the spatial structure requirements of bone tissue regeneration.^31,32 In the present work, the interconnected porous akermanite scaffolds reinforced with nano-TiO₂ were successfully fabricated by SLS. The fracture toughness, Vickers hardness, compressive strength and stiffness were measured as a function of nano-TiO₂. The effects of nano-TiO₂ on the mechanical properties were investigated and discussed. The toughening and strengthening mechanisms of nano-TiO₂ reinforced porous akermanite scaffolds were analyzed. In addition, the bioactivity and cytocompatibility of the akermanite scaffolds were evaluated.

2 Materials and methods

2.1 Materials

The starting akermanite powder (purity ≥ 98%) was obtained from Kunshan Chinese Technology New Materials Co. Ltd. The akermanite powder was synthesized by a sol–gel method using tetraethyl silicate (C₈H₂₀O₄Si), magnesium nitrate (Mg(NO₃)₂·6H₂O) and calcium nitrate (Ca(NO₃)₂·4H₂O). The starting nano-TiO₂ powder (rutile) (purity ≥ 99.9%) was acquired from Nanjing Emperor Nano Material Co. Ltd. Proportions with weight fractions ranging from 0 to 8 wt% of nano-TiO₂ powder were added to the starting akermanite powder. The mixture was firstly milled in an agate mortar for 1 h, and then appropriate amounts of ethanol were added followed by ball milling for 8 h. The SEM micrographs of akermanite with different contents of nano-TiO₂ after mechanical mixing were shown in Fig. 1. The nano-TiO₂ particles were uniform distribution in the akermanite matrix when the contents of nano-TiO₂ were 1 wt% and 5 wt% (Fig. 1a and b). The nano-TiO₂ particles did not disperse well with a little agglomeration when the content of nano-TiO₂ was 8 wt% (Fig. 1c). Previous studies have shown that the agglomeration of nano-particles led to a significant decrease in mechanical properties.^33–35 Therefore, in this paper, the akermanite powder was mixed with different proportions of 0 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt% and 7 wt% of nano-TiO₂ powder to form eight akermanite based composite powder.

Fig. 1 SEM micrographs of the akermanite/nano-TiO₂ after mixing (a) akermanite + 1 wt% nano-TiO₂, (b) akermanite + 5 wt% nano-TiO₂, (c) akermanite + 8 wt% nano-TiO₂.

2.2 Scaffold design and fabrication

A porous scaffold model (L × W × H = 14 × 14 × 5 mm³) with the 3D interconnected pore structure was designed using SolidWorks software (Solidworks 2009, Concord, MA), as shown in Fig. 2a and b. When a porous scaffold was fabricated, the design model was exported in the STL format and then the computer sliced the model into a stack of thin slices. The laser sintering experimental was carried out on a homemade SLS system (a detail description of the sintering system and the parameters are explained in the reference).³⁶ Firstly, a ceramic substrate was placed on the sintering platform. And then, a uniform thin layer of powder was deposited on the substrate, with the layer thickness of 0.1–0.2 mm. Laser was guided to sinter the powder selectively to form a layer. The following processing parameters were used: spot size of 1.2 mm, laser power of 7.8 W, laser scanning speed of 100 mm min⁻¹, and scan line spacing of 2.7 mm. The motion platform changes the sintering direction to y-axis after the completion of the sintering for a strut along x-axis. The similar process was repeated many times until the scaffold was completed. Last, the excess powder surrounding the scaffold was brushed off. The 3D porous scaffold is shown in Fig. 2c and d. It can be seen that the scaffold has an open, uniform and interconnected porous structure.

Fig. 2 (a) Top plane view and (b) cross-section view of the 3D structures of scaffold model: d₁ = 2.7 mm; d₂ = 1.5 mm; d₃ = 14.7 mm; d₄ = 5 mm, (c) top plane view and (d) cross-section view of the fabricated porous scaffold.

2.3 Characterization

The starting powder, the 3D porous scaffold, the Vickers hardness indentation, the grains and their boundaries and the crack were observed by scanning electron microscopy (SEM, JSM-5600LV, Japan). The phase compositions of the starting powders, the composite powders, the fabricated scaffolds and the surface deposition after soaking in SBF were determined by X-ray diffraction (XRD, D8-ADVANCE Bruker AXS Inc., Germany), using Cu-Kα radiation at 40 mA and 40 kV. Data were recorded in the 2θ range of 20–50° (step size 0.02° and scanning rate 8° min⁻¹). The obtained experimental patterns were compared to the standards joint committee on powder diffraction and standards (JCDPS), which involved card # 09-0432 for HAP, # 87-0052 for akermanite and # 76-0317 for rutile TiO₂. Scaffold specimens were polished with diamond pastes down to 1.5 μm and subsequently etched by immersing in a 2 wt% hydrofluoric acid (HF) solution for 30 s to delineate the grain boundary. Each specimen was thoroughly washed with distilled water and rinsed to remove any residual acid after etching. The specimens were dried at room temperature, gold coated, and then examined under high vacuum. The chemical composition of the starting powder and the specimens after etching was evaluated by energy dispersive X-ray spectroscopy (EDS) analysis together with SEM.

Both Vickers hardness and fracture toughness were measured with a Vickers Microindenter (Digital Micro Hardness Tester, HXD-1000 TM/LCD, Shanghai Taiming Optical Instrument Co. Ltd). Prior to indentation, the struts specimens (1 × 1.2 × 1.2 mm³) were mounted in a resin and their surfaces were ground and polished. The indentation tests were performed at a load of 300 gf and a hold of 15 s. The Vickers hardness and fracture toughness were calculated from eqn (1) (ref. 37) and (2) (ref. 38), respectively.

HV = 0.1891F/d² (1)

K_IC = 0.0824F/C^3/2 (2)

where HV is the Vickers hardness (GPa), K_IC is the fracture toughness (MPa m^1/2), F is the applied load (MN), d is the average diagonal line length of the indentation (m), and C the radial crack length (m) measured from the center point of the indentation impression.

The compressive strength and stiffness of the scaffolds (14 × 14 × 5 mm³) were carried out using microcomputer control electronic universal test machine (WD-D1, Shanghai zhuoji instruments Co. Ltd). The crosshead speed was set at 0.5 mm min⁻¹, and the load was axially applied until the scaffold was failed. During the compressive test, the load and displacement were monitored and recorded. Five specimens were prepared from individual compositions for each type of mechanical properties test. Then the average value was taken and the maximum error obtained was found to be less than 5%.

2.4 Apatite-formation ability of the scaffolds in SBF

The bioactivity of the nano-TiO₂ reinforced akermanite scaffolds (14 × 14 × 5 mm³) was investigated by immersion in SBF solutions at 37 °C for 2 and 6 days with the following ionic concentrations (in units of mM): 142.0 Na⁺, 5.0 K⁺, 1.5 Mg²⁺, 2.5 Ca²⁺, 147.8 Cl⁻, 4.2 HCO₃⁻, 1.0 HPO₄²⁻ and 0.5 SO₄²⁻. The solutions were buffered at pH = 7.40 with tris-hydroxymethyl-amminomethane [(CH₂OH)₃CNH₂] and hydrochloric acid [HCl] according to the procedure described by Kokubo et al.³⁹ After the set soaking time, the scaffolds were removed from SBF, gently rinsed with distilled water, and then dried at room temperature in a drying oven for 5 h before further characterization. The surfaces morphology of the scaffolds was observed using SEM and the formation of bone like apatite on the surface of the scaffolds was characterized by EDS.

2.5 Cell attachment and morphology of MG-63 on the scaffolds

The human osteoblast-like MG-63 cells were obtained from American Type Culture Collection (Rockville, MD, USA) and cultured as previously described.⁴⁰ The proliferation medium contained 500 ml MG-63 cells basal medium (DMEM; Cell-gro by Mediatech, Inc., Manassas, VA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA) and 1% penicillin/streptomycin (Gibco). Cells were cultured in a humidified 37 °C/5% CO₂ incubator and the culture medium was changed every 2 days. The morphology of primary MG-63 cells was observed using a light microscope (Olympus IX51, Japan). 4 × 10⁵ cells were added to the porous scaffolds (14 × 14 × 5 mm³) in 96 well plates.

For the measurement of cell adhesion, the number of living cells was measured according to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. After 3, 7, and 10 days cell incubation on the pure akermanite scaffold and the 5 wt% nano-TiO₂ reinforced akermanite scaffold, non-adherent cells were removed by twice gentle washes in PBS. About 1 ml of 16% MTT in DMEM medium was added to each well and incubated at 37 °C in a 5% CO₂ incubator for 4 h. Then 100 μl of the homogenized solution was transferred to the 96 well plates and the optical density was recorded at 570 nm using an enzyme immunosorbent assay reader. By preparing a standard curve for MTT reduction against the cell number, optical-density values can be converted into actual cell numbers. For morphological observation, the scaffolds were removed form the culture wells, rinsed in PBS, and then fixed with 2.5% glutaraldehyde in PBS for 1 h. Subsequently, the scaffolds were dehydrated through a series of graded ethanol alcohols and dried with hexamethyldisilazane at room temperature. The scaffolds were coated with a very thin layer of gold and observed by SEM.

3 Results and discussion

3.1 Porous akermanite scaffolds

The overview of the scaffolds has a well ordered and interconnected pore channel structure (Fig. 3a). The actual size of the struts is 1.2 ± 0.12 mm, while the designed dimension of the struts is 1.2 mm. The reason for this little offset is because some powders in the design pore area are partially fused together. The pore size of the scaffold is around 1.5 mm (Fig. 3b). It has been reported that the better osteogenesis for implants when using pores greater than 300 μm, due to enhanced new bone formation and the formation of capillaries.⁴¹ Hollister et al.⁴² have carried out in vivo studies on hydroxyapatite scaffolds with pore diameters ranging between 400 and 1200 μm in a minipig mandibular defect model. They found significant bone growth on designed scaffolds for all pores, with no statistical difference between pore sizes. Roy et al.⁴³ also found that there was no significant difference in bone growth for 500 and 1600 μm pores for poly (DL-lactic-co-glycolic acid) (PLGA) scaffolds made by a 3D printing technique. There is good bonding between the adjacent layers of the strut (Fig. 3c). This structural characteristic is desirable for improving the mechanical properties of the scaffold. The strut has a very smooth and dense surface (Fig. 3d). It can be inferred that SLS technique can be used to fabricate the scaffold with 3D interconnected pore structures and the laser energy is enough to melt the composite powder with the predetermined process parameters.

Fig. 3 (a) Overview of the pore channels, (b) a single pore channel, (c) the staggered layer structure, (d) the strut surface.

The XRD pattern of the starting powders, the akermanite scaffold with and without TiO₂ was shown in Fig. 4. There were presented the characteristic peaks of akermanite and rutile TiO₂, and no additional phases were detected. The characteristic peaks of TiO₂ appeared in the composite powders and the 7 wt% nano-TiO₂ reinforced akermanite scaffold. All this meant that there were no phase change and reaction between akermanite and TiO₂ after laser sintering.

Fig. 4 XRD pattern of (a) the starting nano-TiO₂ powders, (b) the starting akermanite powders, (c) the akermanite + 7 wt% nano-TiO₂ composite powders, (d) the pure akermanite scaffold and (e) the 7 wt% nano-TiO₂ reinforced akermanite scaffold.

3.2 Mechanical properties of the scaffolds

A representative Vickers indentation on the surface of strut is shown in Fig. 5a. The Vickers indentation diagonals and cracks at the tips of the indentation can be seen clearly. The cracks emanate from the four vertices along the indentation diagonals. The Vickers hardness and fracture toughness are determined by measuring the length of the diagonals and the cracks, respectively. The typical stress–strain curves of the 5 wt% nano-TiO₂ reinforced porous akermanite scaffold are shown in Fig. 5b. The analysis of the stress and strain curves indicates that all scaffolds present a brittle behavior, which is characteristic for ceramic materials. The stiffness is determined by the slope of the initial linear section of the stress–strain curve. The average result of five compressive tests carried out under the same conditions for each type of scaffold is obtained.

Fig. 5 (a) Representative Vickers indentation obtained on the surface of strut, (b) typical stress–strain curve of the 5 wt% nano-TiO₂ reinforced porous akermanite scaffold.

Mechanical properties of the scaffolds in terms of fracture toughness, Vickers hardness, compressive strength and stiffness were investigated. The fracture toughness and Vickers hardness of the scaffolds as a function of nano-TiO₂ are presented in Fig. 6a. It could be seen that the fracture toughness was improved considerably by the addition of nano-TiO₂. A maximum fracture toughness of 2.32 MPa m^1/2 was achieved for the scaffold with 7 wt% nano-TiO₂ content, which is much higher than that of the pure akermanite scaffold (1.82 MPa m^1/2). The Vickers hardness of the pure akermanite scaffold is 5.82 GPa, and it increases to 7.66 GPa when the content of nano-TiO₂ is 7 wt%. The compressive strength and stiffness of scaffolds as a function of nano-TiO₂ are shown in Fig. 6b. It can be observed that the compressive strength and stiffness increase from 3.53 MPa to 22.86 MPa and 31.09 MPa to 122.36 MPa with increasing nano-TiO₂ from 0 to 7 wt%, respectively. The mechanical properties of the scaffolds could be significantly improved by addition of nano-TiO₂.

Fig. 6 (a) Effect of nano-TiO₂ on the fracture toughness and Vickers hardness of scaffolds, (b) effect of nano-TiO₂ on the compressive strength and stiffness of scaffolds.

The mechanical properties increased rapidly with increasing nano-TiO₂ addition from 0 wt% to 5 wt%, while it increased slowly with further increase in nano-TiO₂ (up to 7 wt%). The slow increase of mechanical properties was mainly due to the nano-TiO₂ powders were not dispersed well in the akermanite matrix when nano-TiO₂ increased from 5 wt% to 7 wt%. Furthermore, the presence of residual internal stresses resulting from the thermal expansion mismatch between akermanite and TiO₂ could also decrease the mechanical properties. Previous studies^44,45 showed that calcium phosphate bioceramics (e.g., HAP and β-TCP) have good biocompatibility with human body, but the low mechanical properties are the main limitation in load-bearing applications. Some reports have showed that the fracture toughness of HAP and β-TCP was 0.8–1.2 and 0.95–1.21 MPa m^1/2, respectively.^46,47 In this study, the fracture toughness of pure akermanite scaffold is 1.82 MPa m^1/2, which is much higher than those of calcium phosphate bioceramics. The mechanical properties of the scaffolds are significantly increased with the content of nano-TiO₂ increasing from 0 to 7 wt%. The highest fracture toughness (2.32 MPa m^1/2) of the reinforced porous akermanite scaffolds is slightly higher than the lowest value of cortical bone (2–12 MPa m^1/2),⁴⁸ and the highest compressive strength (22.86 MPa) is much lower than cortical bone (130–180 MPa).⁴⁹ Previous study showed that the fracture toughness of akermanite ceramics is 1.83 MPa m^1/2 sintered at 1370 °C for 6 h,¹⁰ and the compressive strength is between 1.13 to 0.53 MPa with the porosity from 63.5 to 90.3%, respectively.⁵⁰

The SEM/EDS analysis of starting akermanite powders and nano-TiO₂ powders are shown in Fig. 7a and b, respectively. The starting akermanite powders have irregular shape and particle size of about 1–5 μm (Fig. 7a). The starting nano-TiO₂ powders are spherical and slightly agglomerated with an average particle size of 20 nm (Fig. 7b). The corresponding EDS analysis shows that Ca, O, Mg and Si are present on the starting akermanite powders, while only Ti and O are present on nano-TiO₂ powders. The SEM/EDS analysis of the polished and chemically etched surface of the pure akermanite and 5 wt% nano-TiO₂ reinforced akermanite are shown in Fig. 7c and d, respectively. The average grain size was determined from SEM images with the linear intercept method. It can be seen that the micro size grains appearance with clear grain boundaries and the average grain size is about 5.3 μm (Fig. 7c). The EDS analysis of the pure akermanite after etching reveals the presence of Ca, O, Mg and Si, agreeing with the elemental composition of akermanite. Some nano-TiO₂ is dispersed within micro-sized akermanite grains, others are found in the intergranular region (Fig. 7d). The average grain size of the reinforced scaffold is about 3.3 μm. It indicates that the nano-TiO₂ can inhibit the grain growth by pinning effect of grain boundaries.⁵¹ The strength of ceramic will increase with decreasing of grain size according to the Hall–Petch relationship. The EDS analysis confirms the presence of nano-TiO₂ in the akermanite grains as well as along the grain boundaries.

Fig. 7 SEM/EDS of (a) the starting akermanite powders, (b) the starting nano-TiO₂ powders, (c) the pure akermanite after etching and (d) the 5 wt% nano-TiO₂ reinforced akermanite after etching.

The location of nano size grains in the micro size grains matrix can be divided into three types according to the classification of Niihara⁵² as illustrated in Fig. 8. In inter or intra type, the nano size grains are dispersed mainly at the grain boundaries of the matrix grains (Fig. 8a) or within the matrix (Fig. 8b). The inter/intra type with nano size grains are distributed both within the micro size grains and along the grain boundaries (Fig. 8c). According to this classification, the microstructure type of the nano-TiO₂ reinforced akermanite is denoted as the inter/intra type. Sawaguchi et al.⁵³ pointed out that the inter/intra type possess the highest toughness than the other two types.

Fig. 8 Three types of nanocomposites: (a) inter type, that is the nano size grains are located at the grain boundaries of micro size grains. (b) Intra type, that is the nano size grains are distributed within the micro size grains. (c) Inter/intra type, that is the nano size grains are distributed at the grain boundaries as well as within the micro size grains.

The Vickers indentation crack propagation in the pure akermanite and nano-TiO₂ reinforced akermanite is shown in Fig. 9. The crack in the pure akermanite propagates relatively straightly (Fig. 9a). However, the crack in the nano-TiO₂ reinforced akermanite propagates flexurally, which inclines and distorts many times (Fig. 9b). The change of crack direction increases the length of crack path and reduces the stress intensity near crack tip.⁵⁴ The crack is propagating mainly around the nano-TiO₂ particles, occasionally end at the hard particle when the crack tip encounters nano-TiO₂ particles (Fig. 9c and d). These processes can absorb additional amounts of fracture energy, which could apparently enhance the fracture toughness of nano-TiO₂ reinforced akermanite. This investigation is also supported by the mechanical properties tests results in Fig. 6. The thermal expansion coefficient of akermanite and TiO₂ are about 32.1 × 10⁻⁶ °C⁻¹ (ref. 55) and 7 × 10⁻⁶ °C⁻¹,⁵⁶ respectively. Residual stress generated around the nano-TiO₂ particles was due to the thermal expansion mismatch when the reinforced akermanite cooled from the high sintering temperature. The large residual stress gradient is located at the interface between relatively hard nano-TiO₂ particles and soft akermanite matrix. When the crack propagating along the boundaries encounters the nano-TiO₂ hard particles, it can be deflected from its original trajectory, where the crack would be impeded by the intragranular particles and deflected again.

Fig. 9 Crack propagation in the (a) pure akermanite and (b–d) nano-TiO₂ reinforced akermanite.

Meanwhile, the internal stress produced by the nano-TiO₂ particles at the grain boundaries tends to alter the crack propagation path from intergranular fracture to transgranular fracture.⁵⁷ The fracture surfaces of akermanites with different nano-TiO₂ are shown in Fig. 10. The fracture mode of the pure akermanite is intergranular fracture (Fig. 10a). It changes from intergranular fracture to more and more transgranular fracture (Fig. 10b–d) with nano-TiO₂ increasing. The fracture mode of akermanites reinforced with 5 wt% nano-TiO₂ was mainly transgranular fracture (Fig. 10d). The transgranular fracture mode significantly helps to strengthen the grain boundaries and resist against crack propagation. It can be concluded that the addition of nano-TiO₂ particles in akermanite matrix could increase the toughness and strength of porous akermanite scaffolds.

Fig. 10 Fracture surfaces of akermanites with different nano-TiO₂ (a) 0 wt%, (b) 1 wt%, (c) 3 wt%, (d) 5 wt%.

3.3 Bioactivity and biocompatibility of the scaffolds

It is known that TiO₂ has been considered as a bioinert ceramic,⁵⁸ so its content in porous scaffold should be as low as possible. As the mechanical properties of 5 wt% nano-TiO₂ reinforced akermanite scaffold was close to that of 7 wt% nano-TiO₂ reinforced akermanite scaffold, we chose the scaffold with 5 wt% nano-TiO₂ to carry out the biological properties tests. The surface morphologies of the 5 wt% nano-TiO₂ reinforced porous akermanite scaffold after immersion in SBF for 2 and 6 days were shown in Fig. 11a, b, d and e, respectively. It could be seen that the surface of the scaffold was partially covered by some particles after 2 days of immersion in SBF (Fig. 11a). The newly formed particles were worm-like (Fig. 11b). The EDS results revealed that there was obvious signal of P element for the mineralized surface of the scaffold. The deposited particles possessed a higher Ca/P ratio (Ca/P = 2.33) than that of HAP (Ca/P = 1.67) (Fig. 10c). The scaffold surface was fully covered by a thick precipitate layer after immersing in SBF for 6 days (Fig. 11d). The formed precipitate had a sponge-like structure (Fig. 11e). The Ca/P ratio of the layer was about 1.75, which was close to that of HAP (Fig. 11f). The XRD patterns of the scaffolds after soaking in SBF for 2 and 6 days were shown in Fig. 11g and h, respectively. The crystalline peak of apatite at 25.7°, 31.8°, 32.7° and 34° were evident in the pattern,⁵⁹ which indicated that akermanite scaffolds induced the apatite formation when soaked in SBF.

Fig. 11 SEM micrographs of the 5 wt% nano-TiO₂ reinforced porous akermanite scaffolds soaked in SBF for 2 days (a and b) and 6 days (d and e); (c and f) the EDS of (b and e) in a selected region, XRD patterns of the scaffolds after soaking in SBF for 2 days (g) and 6 days (h).

The cross-sectional views of the akermanite scaffold with and without the 5 wt% nano-TiO₂ after soaking in SBF for 2 and 6 days were shown in Fig. 12. It could be seen that the all the scaffolds surfaces were covered by the homogenous three-dimensional (3D) lamellar structure, which was the bone like apatite layer. The thickness of the apatite layer increased obviously with the increase of soaking time. No significant differences were observed in the cross-sectional of the akermanite scaffold with and without 5 wt% nano-TiO₂ after soaking in SBF for the same time. The results indicated that the nano-TiO₂ did not alter the apatite forming ability of akermanite scaffolds. Previous studies^60,61 have shown that the bone like apatite plays an important role in inducing bone formation and enhancing cell adhesion and proliferation. The formation of bone like apatite suggested that the nano-TiO₂ reinforced akermanite porous scaffold possessed good bioactivity.

Fig. 12 Cross-sectional views observed by SEM of the pure akermanite scaffold (a and b) and the 5 wt% nano-TiO₂ reinforced akermanite scaffold (c and d) soaked in SBF for 2 days (a and c) and 6 days (b and d).

The cell viability of MG-63 cells cultured on the akermanite scaffolds with and without the 5 wt% nano-TiO₂ after 3, 7, and 10 days were shown in Fig. 13. Obvious cell proliferation was observed on all of the scaffolds, indicating that all the materials were biocompatible and could support cell proliferation. The viability of MG-63 cells on the scaffolds was statistically lower at the early days but increased dramatically with prolonging the culture time. MG-63 cells proliferated more favorably in the akermanite scaffolds with the 5 wt% nano-TiO₂ than the scaffolds made of akermanite only, which indicated that the addition of 5 wt% nano-TiO₂ could improve the biocompatible of akermanite scaffolds. Studies had shown that TiO₂ was able to enhance osteoblast adhesion and could induce cell growth.^62–64

Fig. 13 MG-63 cells numbers based on an MTT assay within the porous akermanite scaffolds. Error bars represent a standard error of mean values where n = 5.

The light-microscopic morphology of original MG-63 cells viewed under light microscope was shown in Fig. 14a. The original MG-63 cells were fusiform or polygon shaped and uniform in size. The morphologies of MG-63 cultured on the pure akermanite scaffold and the 5 wt% nano-TiO₂ reinforced porous akermanite scaffold for different days were shown in Fig. 14b–d and e–g, respectively. The MG-63 cells attached and spread well on the surface of the scaffold after culturing for 3 days (Fig. 14b and e). The number of MG-63 cells attached on the scaffold surface increased significantly with increasing of culture time (Fig. 14c–d and f–g). The cells were flattened and attached tightly on the scaffolds surfaces with their filopodium and lamellipodium, suggesting good cell viability on all akermanite scaffold. Previous studies⁵⁰ showed that the Ca, Si and Mg ions from akermanite scaffolds dissolution could stimulate MG-63 cells proliferation and this stimulatory effect might be considered as one of the evaluation criteria for bioactivity of the scaffolds. Our results indicated that the nano-TiO₂ reinforced akermanite scaffold possessed excellent biocompatibility to enhance cell attachment and growth.

Fig. 14 Light-microscopic morphology of original MG-63 cells (a); SEM micrographs of the MG-63 cells (arrows) on the pure akermanite scaffold (b–d) and the 5 wt% nano-TiO₂ reinforced akermanite scaffold (e–g) after cell culture for 3 days (b and e), 7 days (c and f) and 10 days (d and g).

4 Conclusions

The nano-TiO₂ reinforced akermanite scaffolds with an open, uniform and interconnected pore structure were successfully fabricated by SLS at the laser power of 7.8 W, laser scan speed of 100 mm min⁻¹ and spot size of 1.2 mm. The results showed that the Vickers hardness, fracture toughness, compressive strength and stiffness increased rapidly from 5.82 GPa to 7.57 GPa, 1.82 to 2.27 MPa m^1/2, 3.53 to 21.81 MPa and 31.09 to 111.05 MPa as nano-TiO₂ increased from 0 to 5 wt%, and then increased slowly to 7.66 GPa, 2.32 MPa m^1/2, 22.86 MPa and 122.36 MPa as nano-TiO₂ increased to 7 wt%, respectively. The mechanisms of remarkable improvement in the mechanical properties were observed to be grain size refinement, crack deflection and fracture mode transition. The akermanite scaffolds reinforced with 5 wt% nano-TiO₂ possessed excellent ability to form bone like apatite in SBF and support cell attachment and growth. It is concluded that the porous akermanite scaffold with 5 wt% of nano-TiO₂ may be a good candidate for bone tissue engineering applications.

Acknowledgements

This work was supported by the following funds: (1) The Natural Science Foundation of China (51222506, 81372366, 81472058); (2) Overseas, Hong Kong & Macao Scholars Collaborated Researching Fund of National Natural Science Foundation of China (81428018); (3) Hunan Provincial Natural Science Foundation of China (14JJ1006); (4) Project supported by the Fok Ying-Tong Education Foundation, China (131050); (5) Shenzhen Strategic Emerging Industrial Development Funds (JCYJ20130401160614372); (6) The Open-End Fund for the Valuable and Precision Instruments of Central South University; (7) The faculty research grant of Central South University (2013JSJJ011, 2013JSJJ046); (8) State Key Laboratory of New Ceramic and Fine Processing Tsinghua University (KF201413); (9) The Fundamental Research Funds for the Central Universities of Central South University.

Notes and references

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