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vivo biocompatibility
Materials Science and Engineering C 78 (2017) 503–512
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Porous Nb-Ti-Ta alloy scaffolds for bone tissue engineering: Fabrication, mechanical properties and in vitro/vivo biocompatibility Jue Liu a, Jianming Ruan a, Lin Chang a, Hailin Yang a,⁎, Wei Ruan b,⁎ a b
State Key Laboratory of Powder Metallurgy, Central South University, Changsha 410083, PR China Department of Anesthesiology, The Second Xiang Ya Hospital, Central South University, Changsha 410011, PR China
a r t i c l e
i n f o
Article history: Received 17 February 2017 Received in revised form 13 April 2017 Accepted 15 April 2017 Available online 19 April 2017 Keywords: Porous materials Alloys Mechanical properties Biocompatibility Pore structure
a b s t r a c t Porous Nb-Ti-Ta (at.%) alloys with the pore size of 100–600 μm and the porosity of 50%–80% were fabricated by the combination of the sponge impregnation technique and sintering method. The results revealed that the pores were well connected with three-dimensional (3D) network structure, which showed morphological similarity to the anisotropic porous structure of human bones. The results also showed that the alloys could provide the compressive Young's modulus of 0.11 ± 0.01 GPa to 2.08 ± 0.09 GPa and the strength of 17.45 ± 2.76 MPa to 121.67 ± 1.76 MPa at different level of porosity, indicating that the mechanical properties of the alloys are similar to those of human bones. Pore structure on the compressive properties was also discussed on the basis of the deformation mode. The relationship between compressive properties and porosity was well consistent with the Gibson-Ashby model. The mechanical properties could be tailored to match different requirements of the human bones. Moreover, the alloys had good biocompatibility due to the porous structure with higher surface, which were suitable for apatite formation and cell adhesion. In conclusion, the porous Nb-Ti-Ta alloy is potentially useful in the hard tissue implants for the appropriate mechanical properties as well as the good biocompatible properties. © 2017 Published by Elsevier B.V.
1. Introduction The appropriate mechanical properties of elastic modulus and compressive stress with good biocompatibility are of great importance to bone substitute materials [1]. A bone-like porous structure is significant to bone tissue engineering application as it can lower the elastic modulus due to its unique geometric architecture, thus reducing the stressshielding effect. Moreover, the 3D porous structure can affect the implants applying in bone tissue engineering in two aspects: Firstly, the above-mentioned structure can promote the ingrowth of the mineralized tissue into the porous network and then make the material firmly anchor to the surrounding bone tissue [2]. Secondly, the different pore size can improve cell adhesion as well as body fluid transport. Therefore, new series of highly porous potential bone graft scaffolds have gained significant attention, such as ceramics and polymer. However, these porous bioactive ceramics and polymers cannot balance the mechanical properties and unique porous structure, which have been seriously restricted their applications [3]. Hence, porous metallic orthopedic implants with proper elastic modulus and load-bearing ability have been widely studied. ⁎ Corresponding authors. E-mail addresses: [email protected] (H. Yang), [email protected] (W. Ruan).
http://dx.doi.org/10.1016/j.msec.2017.04.088 0928-4931/© 2017 Published by Elsevier B.V.
Of all the metallic biomaterials, Ti-based alloys have been important biomaterials for a long time due to several advantages in high specific strength, excellent corrosion resistance and are capable of providing good biocompatibility [4–6]. However, the mismatch between the solid titanium and the surrounding natural bones may lead to bone resorption and implant loosening attributed to “stress shielding effect”. Nb, Ta, Zr and Mo are strong β-stabilizers in Ti alloys, contributing to decreasing the Young's modulus. M. Fischer et al. [7] investigated the microstructural and mechanical properties of a Ti-Nb alloy fabricated by selective laser melting on powder bed of a mixture of Ti and Nb elemental powders (26 at.%). Results showed that laser energy had a significant effect on the compactness and homogeneity of the manufactured parts. V. Sheremetyev et al. [8] investigated the functional fatigue behavior of Ti-22Nb-6Zr (at.%) alloy for load-bearing biomedical applications. The polygonized nanosubgrained (NSS) alloy obtained after cold-rolling with 0.3 true strain and post-deformation annealing at 600 °C showed a low Young's modulus and globally superior fatigue performance due to the involvement of reversible stress-induced martensitic transformation in the deformation process. Moreover, C. Aguilar et al. [9] fabricated the Ti-34Nb-29Ta-xMn (x: 2, 4 and 6 wt% Mn) foams through powder metallurgy at 1300 °C for 3 h using ammonium hydrogen carbonate as a space holder. Measured elastic moduli of the as-sintered foams were around 30 GPa and the values lied in the upper range of elastic modulus of cortical bones. There have been a number of other Ti-based β-type
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Fig. 1. (a)–(d) Porous Nb-Ti-Ta alloys with different porosities; (e)-(h) SEM images showing the morphologies of porous Nb-Ti-Ta alloys.
systems fabricated so far, for example, Ti-Fe-Nb [10], Ti-7Nb-10Mo [11], Ti-31Nb-6Zr-5Mo [12] and Ti-Nb-Zr-Ta-Si-Fe [13]. For porous metallic biomaterials, it is well-known that porosity level and pore sizes are two important aspects that can significantly influence the mechanical behavior and biocompatibility. Gibson-Ashby model has concluded that Young's modulus and yield stress are substantially affected by porosity [14]. Therefore, it is important to design the pore structure using a proper fabrication process, which can effectively moderate the mechanical properties and reduce stress-shielding effect. Regarding the fabrication process of porous metallic biomaterials, various fabrication methods have been proposed, including powder sintering [15,16], freeze casting [17,18], space holder method [15,16] and rapid prototyping [19,20]. However, the poor mechanical properties and high defects such as impurities have restricted their applications. Up to now, several new fabrication processes have been proposed to prepare porous alloys. Porous titanium with an average pore size of 100–650 μm and porosity of 30–70% was fabricated by diffusion bonding of titanium meshes, whose compressive Young's modulus and yield stress were in the range of 1–7.5 GPa and 10–110 MPa, respectively [21]. Though it can be capable of providing suitable mechanical properties, it is difficult to fabricate porous titanium possessing an isotropic structure with square pores to adapt to the complex human body environment and anisotropic porous structure of bones. Titanium hydride (TiH2) has been widely used in preparation of porous alloys as a conventional pore forming active agent [2]. However, it is difficult to
Table 1 Porosity of Nb-Ti-Ta alloys. Weight percent of PVA (wt%)
Slurry-powder Porosity(%) ratio (g/ml) (mass-volume method)
Porosity(%) (Archimedes principle)
Open porosity(%)
3 3 5 7
3 4.5 4.5 6
81.35 ± 1.05 72.88 ± 2.03 64.36 ± 1.91 54.15 ± 0.89
95.0 ± 1.4 94.4 ± 1.8 93.6 ± 2.1 90.5 ± 1.1
82.83 ± 0.95 71.40 ± 0.87 62.60 ± 1.46 55.57 ± 0.27
possess excellent design and accurate control of the porosity for a space-holder method. In this paper, porous Nb-Ti-Ta alloys with different porosities were introduced using the sponge impregnation technique and the powder metallurgy (P/M) method in combination. The physical (pore structure and mechanical properties) and chemical (phase and hydroxyapatite formation) properties of these scaffolds were systematically investigated. Additionally, the biocompatibility of porous Nb-Ti-Ta alloy scaffolds was studied by culturing MG-63 cell in vitro and a rabbit model was used to analyze the osteogenic ability in vivo.
2. Materials and methods 2.1. Fabrication of porous Nb-Ti-Ta alloy scaffolds The starting elemental metal powders of Nb (Purity ≥ 99.5%,average particle size 40.52 μm), Ta (Purity ≥ 99.6%,average particle size 8.45 μm) and Ti (Purity ≥ 99.6%,average particle size 18.34 μm) were weighted with a nominal composition of Nb-Ti-Ta (at.%). These elemental powders were blended for 4 h in a roller mixer. A commercial polyurethane sponge was employed to introduce macroporosity and shaped to a component with the size of Φ10 × 20 mm. Before being impregnated, the desired shape and size polyurethane foam templates were firstly treated with following processes: (i) pretreated by washing in 10% NaOH solution at the temperature of 40–60 °C for 20 min; (ii) cleaned with deionized water repeatedly; (iii) dried in a furnace at 35 °C for 24 h. In this method, polyving akohol (PVA) aqueous solution was used as a binder. In order to control the porosity and pore size, the slurry viscosity and slurry-powder (SP) ratio should be well controlled. The slurry viscosity was defined as the weight percent of PVA and SP ratio was calculated according to the following equation:
SP ¼
Mpowder VPVA
ð1Þ
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Fig. 2. XRD spectrum of the porous Nb-Ti-Ta alloy with 60% porosity.
where M is the mass of corresponding powder (g) and V is the volume of PVA solution (ml). The mixed powder slurries were mainatined under constant magnetic stirring for 1 h, and then the sponges were carefully impregnated in the slurries. Subsequently, the specimens were dried at 40 °C for 24 h and heated at 400 °C for 2 h under a vacuum to burn out the sponge scaffolds. Subsequently the sintering process was carried out in high vacuum (≤9 × 10−3 Pa) at 1700 °C for 2 h with the heating rate of 10 °C/min and then furnace-cooled to room temperature.
2.2. Characterization of porous Nb-Ti-Ta alloy scaffolds The phase composition and morphology analysis were examined by X-ray diffraction (XRD, D/Max-2550, Japan) and A NOVATM Nano 230 scanning electron microscope (SEM) with an energy dispersive X-ray (EDX) unit. The apparent porosities of the scaffolds were measured by both mass-volume method and Archimedes principle, which was described as elsewhere [4,22]. Compression tests were carried out with a crosshead speed of 1 mm/min at room temperature by using an Instron
Fig. 3. Relationship between pore size distribution and porosity for porous Nb-Ti-Ta alloys: (a) P = 50%, (b) P = 60%, (c) P = 70% and (d) P = 80%. (e) Relationship between average pore size and porosity for porous Nb-Ti-Ta alloys.
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phosphate. Alloys were sterilized with autoclave at 120 °C for 30 min, then incubated with 1 ml culture medium containing 5 × 104 cells in a humidified atmosphere with 5% CO2 for 4 h. All the samples with cells were transferred into 24-well plates so that the attachment could happen. After the desired time (6 h, 48 h and 7 days) culture, the media were removed and washed by phoaphate buffered saline (PBS), then pre-fixed in 3% glutaraldehyde for 30 min. Gradient ethanol dehydration and subsequent gradient hexamethl-disilazane dehydration were followed. Cellular attachment, spreading behavior and detailed morphologies of as-obtained specimens could be observed by SEM after Au sputtering. 2.4. In vivo test in a rabbit model
Fig. 4. The compressive strength and elastic modulus of porous Nb-Ti-Ta alloys with different porosities and pure Nb, Ta [24] made by the same process with 60% porosity. For comparison, the corresponding parameters of cancellous bone and cortical bone are listed from Ref. [24].
3369 maching. The slope of the initial linear portion of the stress-strain curve was used to define the elastic modulus and the compressive strength. At least five parallel tests were conducted for each group to ensure the reproducibility. 2.3. Apatite-forming ability and in vitro experiments 2.3.1. Apatite formation Apatite-forming test was carried out by immersing the samples into SBF solution (placed in 100 ml conical flask) at 37 °C for different time (3 days, 9 days and 14 days) and the SBF solution was refreshed every 2 days. After being immersed, samples were taken out and cleaned with distilled water, then dried in an oven at 40 °C for further characterization. 2.3.2. Cell adhesion and proliferation Human osteoblast-like MG-63 cells were seeded and cultured with medium consisting of RPMI 1640 supplement with 10% (v/v) fetal bovine serum (FBS, Thero), 1% antibiotics and 0.85 mM ascorbic acid-2
Fig. 5. SEM images showing the microstructure of sintering neck of porous Nb-Ti-Ta alloy.
The animal experimentation protocol was submitted and approved by local animal care and safety committee. The experiments were performed on adult New Zealand white rabbits weighting 2.5–3.0 kg. The rabbits were kept individually in stainless steel cages, which provided standard food and sterilized drinking water with unrestricted mobility and were checked daily. All animals were operated under general anesthesia. The rabbits were executed after 12 weeks, and the implant-bone compounds were harvested. All samples were dried in an oven and the detailed morphologies of as-obtained specimens could be observed by SEM after Au sputtering. 3. Results 3.1. Pore structure Porous Nb-Ti-Ta alloys with different porosities are shown a good 3D interconnected network and the anisotropic porous structure was very similar to that of trabecular bone, especially for the case with a higher porosity level of 60%–80%. As shown in Fig. 1, with the decrease of porosity, the interconnectivity became poorer. The pore connectivity was crucial for transformation of body fluids when the scaffolds were implanted into human body and it was also of great importance during surface modifications for chemical solutions throughout the pores. The pore connectivity was mainly achieved from the macro pores, which resulted from the macroporosity of sponge scaffolds and micro pores were formed by the evaporation of PVA and gradual elimination of the sponge scaffolds during the following sintering process. Previous investigations [1,4] have demonstrated that equiaxed pores were less efficient than the anisotropic porous structure, thus it indicate that the present porous Nb-Ti-Ta alloys possessed the anisometric pore structure, which was suitable for bone implants and could reduce “stress shielding” effect. Table 1 displays the porosities measured by Achimedes principle in association with the calculated values by mass-volume method for porous Nb-Ti-Ta alloys. It was obvious that the difference between the measured and calculated data was b3%, indicating that the above-mentioned process could excellently control the porosity. XRD spectrum (Fig. 2) of the porous alloys only consisted of β phase. No hydride, oxide or other impurities were detected in the XRD patterns. It indicates that the manufacturing process did not introduce any other impurities, which have detrimental effects on the mechanical properties and biocompatibility of the materials. Quantitatively, pore size distribution of the porous Nb-Ti-Ta alloys in terms of the mean diameter using image analysis method based on the SEM image was shown in Fig. 3, in which hundreds of pores were measured to confirm the average pore size. For the alloy scaffold with 50% porosity, pore size was relatively homogenous and N85% pore sizes were in the range of 50–300 μm and none of pores was larger than 400 μm. With the increase in porosity, the pore size distribution became wider and the maximum pore size became bigger. As the porosity level reached to 70%, almost 90% pores were in the range of 150–400 μm, as shown in Fig. 3(a)-(d). Furthermore, Fig. 3(e) exhibited the relationship between pore size and porosity for the porous alloy scaffolds. The
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Fig. 6. SEM images showing the microstructure of porous Nb-Ti-Ta alloys after immersion in SBF for (a) 3 days, (b) 9 days and (c) 14 days.
average pore size of porous alloy scaffold with 50% porosity was 199 μm, and the average pore size increased to 317 μm when porosity reached to 80%. Qualitatively, porosity, pore size and its distribution of porous NbTi-Ta alloy scaffolds could be tailored in the present fabrication process. As the pore size considerably influenced the biocompatibility of porous implants [7] and pore size in the range of 100–600 μm was well suited for the ingrowth of new bone tissues [1,7], the present porous Nb-TiTa alloy scaffolds could have the capability of matching different requirements of the human bones. 3.2. Mechanical properties In the present work, the relationship between the elastic modulus and the compressive strength is shown in Fig. 4. Both the modulus and the strength decreased with the increase of porosity. The compressive strength and Young's modulus of the porous Nb-Ti-Ta alloy with different porosities, achieved in the range of from 17.45 ± 2.76 MPa to 121.67 ± 1.76 MPa and 0.11 ± 0.01 GPa to 2.08 ± 0.09 GPa. Young's modulus and compressive strength of human trabecular bones are in the range of 0.1–30 GPa and 2–70 MPa with different values depending on age and experimental conditions [23]. Metallic biomaterials for medical applications are suffering from the “stress shielding” effect, which could lead to bone resorption and implant loosening. Therefore, the elastic modulus and compressive strength of medical implants should be well tailored to match different requirements of the human bones and controlled in the range of 0.1–30 GPa and around 100 MPa, respectively [24]. It should be noted that the developed porous Nb-Ti-Ta alloys processed the optimum compressive strength around 100 MPa, which was in the range of between cancellous bone and cortical bone, and could meet the mechanical properties of trabecular bone well. Moreover, the
Young's modulus of the porous scaffolds was falling in the range of between cancellous bone and cortical bone, which had the potential to minimize the stress shielding effect. As well known, it is hard to obtain a good balance between suitable modulus and enough compressive strength for porous scaffolds. The SEM micrograph (Fig. 5) shows the morphology of sintering neck between particles formed during diffusion bonding, which would strengthen the bond between particles and lead to the shrinkage of the micropores, thus offering a balance between the high porosity and enough load bearing ability. It is also obvious that there are no unmolten powders and impurities on the surface of porous structure, which may present in the samples fabricated by a rapid prototyping and a space-holder method and could show detrimental effects on the mechanical properties [7]. 3.3. In vitro studies with SBF and cell adhesion Stimulated body fluid (SBF) was used to evaluate the apatiteforming ability of the obtained alloys, and the materials can achieve the bone-bonding process via the induced apatite formation in body fluids [25]. Typically, Fig. 6 compares the morphologies Nb-Ti-Ta alloy scaffolds after immersion in SBF for 3 days, 9 days and 14 days. It was found that all the surfaces induced nucleation of particles after immersion in SBF, the density of the particles in the scaffolds increased with immersing time. Fig. 6 (c) presents that the precipitated particles became coarser and homogenously with dome shaped particles after 14 days immersion in SBF. It should be noted that the rough porous surface may act as better sites for the nucleation and growth of particles, which benefits the formation of apatite. Furthermore, to evaluate the in vitro biocompatibility, the adhesion and proliferation of MG-63 cells on the porous Nb-Ti-Ta alloy scaffolds is shown in Fig.7(a)(b). It can be observed that the elongated and
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Fig. 7. SEM micrographs showing the surface morphologies of Nb-Ti-Ta alloys after MG-63 cell cultured for (a) 6 h, (b) 48 h and (c) 7 days.
flattened MG-63 cells have formed numerous filopodia and lamellipodia, anchoring on the surface and into the internal surface of the pores. Qualitatively, after 7 days culture (Fig. 7(c)), cells could spread well and established connection with neighbor cells, suggesting that the porous alloy scaffolds could offer a good/excellent adhesion and proliferation ability. 3.4. In vivo test The SEM images showed the microstructure of porous Nb-Ti-Ta alloys after implanted on rabbit model for 12 weeks, as shown in Fig. 8. After 12 weeks implantation, the original porous scaffolds were covered with newly born bone tissues, which showed no obvious gap. From the high magnification image on an area of interest, the bone-trabecular like areas confirmed the new bone tissue generation, as shown in Fig. 8(b). Furthermore, the Ca/P ratio of the formed bone tisssue were detected by EDS, and the result fixed at 1.25, which was lower than that of natural HA of 1.67. 4. Discussion 4.1. Structure of porous alloy scaffolds Up to now, various fabrication processes have been proposed to prepare porous alloys with elongated pores, which only possess equiaxed pores [2]. Porous alloys with equiaxed pores are often manufactured by simply controlling the aspect ratio and the amount of pore-foaming agent which is difficult in reducing “stress shielding” effect and could hardly adapt to the complex human body environment. For porous materials with potential for trabecular bone implant applications, the structure of pores is a key factor that can affect long term
fixation. In order to further reveal the porous structure of Nb-Ti-Ta alloys fabricated by the combination of the sponge impregnation technique and sintering method, Fig. 9 presents schematic diagrams of the fabrication methods for some widely used metallic implants. From Fig. 9(a), for porous titanium fabricated by diffusion bonding of titanium meshes, the porosity and pore size could be controlled by adjusting the aspect ratio of titanium meshes. Therefore, the pore was regular and exhibited a latitude-longitude like structure, which could be difficult to adapt to the complex structure of human bones. From Fig. 9(b), porous Ti-18 at.% Nb-4 at.% Sn alloy was prepared by a space-holder method using NH4HCO3 as space-holder. However, it is difficult to possess porous alloys with excellent design and accurate control of the porosity by simply adjusting the amount of space-holder in abovementioned method [26,27]. For the present fabrication method of porous alloy scaffolds, the sponge scaffolds were impregnated into the mixed powder slurries during the process of impregnation. Simultaneously, the skin of the pores delivered the mixed powder slurries to different inter-connected surface of pores due to the drive of both extrusion force and capillary force. Subsequently, in the following dry process, the water in slurry was evaporated; the powder particles were bonded together on the internal surface of the pores of the sponge scaffolds. Finally, in the sintering process with high vacuum, the pores size came to shrink through gradual elimination of the sponge scaffolds and thus achieved the formation of the 3D porous structure with elongated pores. As apparently seen in Fig. 10, the present porous Nb-Ti-Ta alloy scaffolds could attract much more attention due to their morphological similarity to the anisotropic porous structure of bones. B. Vafaeian et al. [28] simulated the porous trabecular bone-mimicking structure based on the micro-scale finite element method (FEM) and both the numerical simulation and experimental results were well agreed with Fig. 10.
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Fig. 8. (a) SEM images showing the morphologies of porous Nb-Ti-Ta alloys after in vivo test with rabbit model for 12 weeks; (b) high magnification of newly born bone tissues and (c) EDS result of newly born bone tissues.
4.2. Effect of porosity on compressive properties Theoretically, the compressive strength and elastic modulus of a porous solid are closely related to the porosity level, distribution and sizes. To design porous scaffolds with proper mechanical properties, the
relationship between compressive properties and porosity should be studied quantitatively. Gibson et al. [14] have demonstrated in detail that compressive properties of porous solids with different porosities, and established equations well known as Gibson-Ashby model to illustrate the relationship. According to Gibson-Ashby model, the
Fig. 9. Outline of fabrication method of porous alloys: (a) diffusion bonding of titanium meshes [21] and (b) space-holder method process [32].
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Fig. 10. (a) Schematic diagrams showing the process of poring by sponge impregnation technique; (b) SEM micrograph showing the morphology of cancellous bone.
relationship between the compressive strength, elastic modulus and porosity can be expressed as follows: E=Es ¼ C1 ðρ=ρs Þn1
ð2Þ
σ pl =σ ys ¼ C2 ðρ=ρs Þn2
ð3Þ
where E, σpl and (ρ/ρs) are Young's modulus, plateau stress and relative density of porous solids, respectively. Es and σys are elastic modulus and yield strength of corresponding metals, respectively. C1 and C2 are constants depending on the porous structure [14]. A regression analysis of the mechanical properties of the porous NbTi-Ta alloys was performed, as shown in Fig. 11. The exponents n1 and n2 were determined to be around 2.0 and 1.3, respectively. It indicated that the relationship between the mechanical properties and the relative densities of the porous alloy scaffolds could conform to the Gibson-Ashby model when C1 = 0.08 and C2 = 0.61. Therefore, the compressive properties of the porous scaffolds fabricated by the present method could be well predicted and the desired mechanical properties could be tailored by porosity to match those of the human bones based on the Gibson-Ashby model.
4.3. Effect of pore on biocompatibility behavior Structurally, several key aspects play important roles for successful porous scaffolds as medical applications. Previous studies [4,29,30] have pointed out that parameters, such as porosity level, pore sizes and morphology, had direct effects on the interaction between implants and their surrounding tissues. Moreover, a large number of research papers have identified that the macropores in the range of 100–600 μm can evidently promote bone tissue ingrowth and eventually achieve good implant fixation [4]. The micropores (5–20 μm) could offer a considerably good fixation of earlier cell attachment due to a bigger surface area and roughness of pore surfaces [4,30–32]. Another important parameter is open porosity. As well known, the better connectivity of porous materials is, the more connected channel for cell ingrowth. Because it is helpful for cells to adhere, proliferate and differentiate as well as offer channels to allow nutrient delivery and waste removal. In the present work, the prepared porous Nb-Ti-Ta alloys showed a 3D interconnected porous structure with the open porosity as high as 90%. The spontaneously formed oxides will provide a bio-inert layer on the alloy surface in the complex human body fluids [4,33]. Therefore, the enrichment of oxides on the surface can suppress the dissolution
Fig. 11. Relationship between compressive properties and relative density for porous Nb-Ti-Ta alloys.
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Fig. 12. Schematic diagrams showing the mechanism of apatite formation on Nb-Ti-Ta alloy in SBF.
of Nb, Ta and Ti ions, which may play a significant role in determining the interaction between implant materials and surrounding tissues. Furthermore, previous studies showed that Ti-OH can be formed in Tibased alloys after SBF immersion [34,35]. As shown in Fig. 12, the mechanism of apatite formation on the Nb-Ti-Ta alloys in SBF was interpreted in terms of electrostatic interactions between the surface and the ions in SBF. Ti-OH as well as Nb-OH [35,36] and Ta-OH [35,37] could be formed on the surface of Nb-Ti-Ta alloys, which is beneficial to the formation of apatite for a long term. Previous studies confirmed that the morphology of cells grown on the surface of alloys could reveal the interactions between the cells and the surface of scaffolds, especially in the early phases of culture [38–40]. As shown in cell attachment and adhesion morphology from the SEM micrographs, MG-63 cells cultured on porous Nb-Ti-Ta alloy scaffolds preferentially presented both lamellipodia and filopodia, anchored on the surfaces and even into the internal surface of the pores. According to the previous results obtained with the cells on dense alloys [41–43], these morphologies reveal more tightly adhering cells. It is known that the in vitro biological test could reflect the early in vivo response, so we can hypothesize that porous Nb-Ti-Ta alloy scaffolds could enhance cellular proliferation by stimulating the transforming growth factor-β (TGF-β) and induce early differentiation via promoting the transcription of alkaline phosphatase (ALP) [44].
5. Conclusion (1) Highly porous Nb-Ti-Ta (at.%) alloys with pore sizes of 100–600 μm and porosity level of 50%–80% were fabricated by the combination of the sponge impregnation technique and sintering method, which were capable of making 3D interconnected anisotropic porous structure. The porous scaffolds provided a compressive Young's modulus of 0.11 ± 0.01 GPa to 2.08 ± 0.09 GPa and the strength of 17.45 ± 2.76 MPa to 121.67 ± 1.76 MPa. (2) The relationship between compressive properties and porosity levels was well consistent with the Gibson-Ashby model and the mechanical properties could be tailored to match different requirements of the human bones. (3) The SBF test revealed that porous Nb-Ti-Ta alloys had the potential to induce apatite in vitro, which was evidently beneficial to the interaction between implant materials and surrounding tissues in human body fluid environment. Porous Nb-Ti-Ta alloys also had an excellent biocompatibility, which could enhance adhesion of MG-63 cells. Moreover, the porous structure could induce cells into the pores, offering good biological fixation and the bone-trabecular like areas from the in vivo test further confirmed the new bone tissue generation.
(4) Porous Nb-Ti-Ta alloys are quite promising as a new candidate of biomedical application after evaluating both the mechanical and biological properties. Acknowledgements The authors would like to acknowledge the National Natural Science Foundation of China (No. 51274247, 51404302) and the Fundamental Research Funds for the Central Universities of Central South University (No. 2016zzts033) for financial support. References [1] Jae-Young Rho, Liisa Kuhn-Spearing, Peter Zioupos, Mechanical properties and the hierarchical structure of bone, Med. Eng. Phys. 20 (1998) 92–102. [2] X. Rao, C.L. Chu, Y.Y. Zheng, Phase composition, microstructure, and mechanical properties of porous Ti-Nb-Zr alloys prepared by a two-step foaming powder metallurgy method, J. Mech. Behav. Biomed. 34 (2014) 27–36. [3] Yao Dai, Hairong Liu, Binbin Liu, Zhenxing Wang, Yongsheng Li, Guangdong Zhou, Porous β-Ca2SiO4 ceramic scaffolds for bone tissue engineering: in vitro and in vivo characterization, Ceram. Int. 41 (2015) 5894–5902. [4] E. Eisenbarth, D. Velten, M. MÜller, R. Thull, J. Breme, Biocompatibility of β-stabilizing elements of titanium alloys, Biomaterials 25 (2004) 5705–5713. [5] Y. Okazaki, E. Gotoh, Metal ion effects on different types of cell line, metal ion incorporation into L929 and MC3T3-E1 cells, and activation of macrophage-like J774.1 cells, Mater. Sci. Eng. C 33 (2013) 1993–2001. [6] Dapeng Zhao, Kebe Chang, Thomas Ebel, Qian Ma, Regine Willumeit, Ming Yan, Florian Pyczak, Microstructure and mechanical behavior of metal injection molded Ti-Nb binary alloys as biomedical material, J. Mech. Behav. Biomed. 28 (2013) 171–182. [7] M. Fischer, D. Joguet, G. Robin, L. Peltier, P. Laheurte, In situ elaboration of a binary Ti-26Nb alloy by selective laser melting of elemental titanium and niobium mixed powders, Mater. Sci. Eng. C 62 (2016) 852–859. [8] V. Sheremetyev, V. Brailovski, S. Prokoshkin, K. Inaekyan, S. Dubinskiy, Functional fatigue behavior of superelastic beta Ti-22Nb-6Zr(at%) alloy for load-bearing biomedical applications, Mater. Sci. Eng. C 58 (2016) 935–944. [9] C. Aguilar, C. Guerra, S. Lascano, D. Guzman, P.A. Rojas, M. Thirumurugan, L. Bejar, A. Medina, Synthesis and characterization of Ti-Ta-Nb-Mn foams, Mater. Sci. Eng. C 58 (2016) 420–431. [10] Shima Ehtemam-Haghighi, Yujing Liu, Guanghui Cao, Lai-Chang Zhang, Influence of Nb on the β → α″ martensitic phase transformation and properties of the newly designed Ti-Fe-Nb alloys, Mater. Sci. Eng. C 60 (2016) 503–510. [11] Ruowei Yi, Huiqun Liu, Danqing Yi, Weifeng Wan, Bin Wang, Yong Jiang, Qi Yang, Dingchun Wang, Qi Gao, Yanfei Xu, Qian Tang, Precipitation hardening and microstructure evolution of the Ti-7Nb-10Mo alloy during aging, Mater. Sci. Eng. C 63 (2016) 577–586. [12] S.X. Liang, X.J. Feng, L.X. Yin, X.Y. Liu, M.Z. Ma, R.P. Liu, Development of a new β Ti alloy with low modulus and favorable plasticity for implant material, Mater. Sci. Eng. C 61 (2016) 338–343. [13] Ivana Kopova, Josef Stráský, Petr Harcuba, Michal Landa, Miloš Janeček, Lucie Bačákova, Newly developed Ti-Nb-Zr-Ta-Si-Fe biomedical beta titanium alloys with increased strength and enhanced biocompatibility, Mater. Sci. Eng. C 60 (2016) 230–238. [14] Lorna J. Gibson, Biomechanics of cellular solids, J. Biomech. 38 (2005) 377–399. [15] S.L. Zhu, X.J. Yang, D.H. Fu, L.Y. Zhang, C.Y. Li, Z.D. Cui, Stress–strain behavior of porous NiTi alloys prepared by powders sintering, Mater. Sci. Eng. C 408 (2005) 264–268.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Porous Nb-Ti-Ta alloy scaffolds for bone tissue engineering: Fabrication, mechanical properties and in vitro/vivo biocompatibility Jue Liu a, Jianming Ruan a, Lin Chang a, Hailin Yang a,⁎, Wei Ruan b,⁎ a b
State Key Laboratory of Powder Metallurgy, Central South University, Changsha 410083, PR China Department of Anesthesiology, The Second Xiang Ya Hospital, Central South University, Changsha 410011, PR China
a r t i c l e
i n f o
Article history: Received 17 February 2017 Received in revised form 13 April 2017 Accepted 15 April 2017 Available online 19 April 2017 Keywords: Porous materials Alloys Mechanical properties Biocompatibility Pore structure
a b s t r a c t Porous Nb-Ti-Ta (at.%) alloys with the pore size of 100–600 μm and the porosity of 50%–80% were fabricated by the combination of the sponge impregnation technique and sintering method. The results revealed that the pores were well connected with three-dimensional (3D) network structure, which showed morphological similarity to the anisotropic porous structure of human bones. The results also showed that the alloys could provide the compressive Young's modulus of 0.11 ± 0.01 GPa to 2.08 ± 0.09 GPa and the strength of 17.45 ± 2.76 MPa to 121.67 ± 1.76 MPa at different level of porosity, indicating that the mechanical properties of the alloys are similar to those of human bones. Pore structure on the compressive properties was also discussed on the basis of the deformation mode. The relationship between compressive properties and porosity was well consistent with the Gibson-Ashby model. The mechanical properties could be tailored to match different requirements of the human bones. Moreover, the alloys had good biocompatibility due to the porous structure with higher surface, which were suitable for apatite formation and cell adhesion. In conclusion, the porous Nb-Ti-Ta alloy is potentially useful in the hard tissue implants for the appropriate mechanical properties as well as the good biocompatible properties. © 2017 Published by Elsevier B.V.
1. Introduction The appropriate mechanical properties of elastic modulus and compressive stress with good biocompatibility are of great importance to bone substitute materials [1]. A bone-like porous structure is significant to bone tissue engineering application as it can lower the elastic modulus due to its unique geometric architecture, thus reducing the stressshielding effect. Moreover, the 3D porous structure can affect the implants applying in bone tissue engineering in two aspects: Firstly, the above-mentioned structure can promote the ingrowth of the mineralized tissue into the porous network and then make the material firmly anchor to the surrounding bone tissue [2]. Secondly, the different pore size can improve cell adhesion as well as body fluid transport. Therefore, new series of highly porous potential bone graft scaffolds have gained significant attention, such as ceramics and polymer. However, these porous bioactive ceramics and polymers cannot balance the mechanical properties and unique porous structure, which have been seriously restricted their applications [3]. Hence, porous metallic orthopedic implants with proper elastic modulus and load-bearing ability have been widely studied. ⁎ Corresponding authors. E-mail addresses: [email protected] (H. Yang), [email protected] (W. Ruan).
http://dx.doi.org/10.1016/j.msec.2017.04.088 0928-4931/© 2017 Published by Elsevier B.V.
Of all the metallic biomaterials, Ti-based alloys have been important biomaterials for a long time due to several advantages in high specific strength, excellent corrosion resistance and are capable of providing good biocompatibility [4–6]. However, the mismatch between the solid titanium and the surrounding natural bones may lead to bone resorption and implant loosening attributed to “stress shielding effect”. Nb, Ta, Zr and Mo are strong β-stabilizers in Ti alloys, contributing to decreasing the Young's modulus. M. Fischer et al. [7] investigated the microstructural and mechanical properties of a Ti-Nb alloy fabricated by selective laser melting on powder bed of a mixture of Ti and Nb elemental powders (26 at.%). Results showed that laser energy had a significant effect on the compactness and homogeneity of the manufactured parts. V. Sheremetyev et al. [8] investigated the functional fatigue behavior of Ti-22Nb-6Zr (at.%) alloy for load-bearing biomedical applications. The polygonized nanosubgrained (NSS) alloy obtained after cold-rolling with 0.3 true strain and post-deformation annealing at 600 °C showed a low Young's modulus and globally superior fatigue performance due to the involvement of reversible stress-induced martensitic transformation in the deformation process. Moreover, C. Aguilar et al. [9] fabricated the Ti-34Nb-29Ta-xMn (x: 2, 4 and 6 wt% Mn) foams through powder metallurgy at 1300 °C for 3 h using ammonium hydrogen carbonate as a space holder. Measured elastic moduli of the as-sintered foams were around 30 GPa and the values lied in the upper range of elastic modulus of cortical bones. There have been a number of other Ti-based β-type
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Fig. 1. (a)–(d) Porous Nb-Ti-Ta alloys with different porosities; (e)-(h) SEM images showing the morphologies of porous Nb-Ti-Ta alloys.
systems fabricated so far, for example, Ti-Fe-Nb [10], Ti-7Nb-10Mo [11], Ti-31Nb-6Zr-5Mo [12] and Ti-Nb-Zr-Ta-Si-Fe [13]. For porous metallic biomaterials, it is well-known that porosity level and pore sizes are two important aspects that can significantly influence the mechanical behavior and biocompatibility. Gibson-Ashby model has concluded that Young's modulus and yield stress are substantially affected by porosity [14]. Therefore, it is important to design the pore structure using a proper fabrication process, which can effectively moderate the mechanical properties and reduce stress-shielding effect. Regarding the fabrication process of porous metallic biomaterials, various fabrication methods have been proposed, including powder sintering [15,16], freeze casting [17,18], space holder method [15,16] and rapid prototyping [19,20]. However, the poor mechanical properties and high defects such as impurities have restricted their applications. Up to now, several new fabrication processes have been proposed to prepare porous alloys. Porous titanium with an average pore size of 100–650 μm and porosity of 30–70% was fabricated by diffusion bonding of titanium meshes, whose compressive Young's modulus and yield stress were in the range of 1–7.5 GPa and 10–110 MPa, respectively [21]. Though it can be capable of providing suitable mechanical properties, it is difficult to fabricate porous titanium possessing an isotropic structure with square pores to adapt to the complex human body environment and anisotropic porous structure of bones. Titanium hydride (TiH2) has been widely used in preparation of porous alloys as a conventional pore forming active agent [2]. However, it is difficult to
Table 1 Porosity of Nb-Ti-Ta alloys. Weight percent of PVA (wt%)
Slurry-powder Porosity(%) ratio (g/ml) (mass-volume method)
Porosity(%) (Archimedes principle)
Open porosity(%)
3 3 5 7
3 4.5 4.5 6
81.35 ± 1.05 72.88 ± 2.03 64.36 ± 1.91 54.15 ± 0.89
95.0 ± 1.4 94.4 ± 1.8 93.6 ± 2.1 90.5 ± 1.1
82.83 ± 0.95 71.40 ± 0.87 62.60 ± 1.46 55.57 ± 0.27
possess excellent design and accurate control of the porosity for a space-holder method. In this paper, porous Nb-Ti-Ta alloys with different porosities were introduced using the sponge impregnation technique and the powder metallurgy (P/M) method in combination. The physical (pore structure and mechanical properties) and chemical (phase and hydroxyapatite formation) properties of these scaffolds were systematically investigated. Additionally, the biocompatibility of porous Nb-Ti-Ta alloy scaffolds was studied by culturing MG-63 cell in vitro and a rabbit model was used to analyze the osteogenic ability in vivo.
2. Materials and methods 2.1. Fabrication of porous Nb-Ti-Ta alloy scaffolds The starting elemental metal powders of Nb (Purity ≥ 99.5%,average particle size 40.52 μm), Ta (Purity ≥ 99.6%,average particle size 8.45 μm) and Ti (Purity ≥ 99.6%,average particle size 18.34 μm) were weighted with a nominal composition of Nb-Ti-Ta (at.%). These elemental powders were blended for 4 h in a roller mixer. A commercial polyurethane sponge was employed to introduce macroporosity and shaped to a component with the size of Φ10 × 20 mm. Before being impregnated, the desired shape and size polyurethane foam templates were firstly treated with following processes: (i) pretreated by washing in 10% NaOH solution at the temperature of 40–60 °C for 20 min; (ii) cleaned with deionized water repeatedly; (iii) dried in a furnace at 35 °C for 24 h. In this method, polyving akohol (PVA) aqueous solution was used as a binder. In order to control the porosity and pore size, the slurry viscosity and slurry-powder (SP) ratio should be well controlled. The slurry viscosity was defined as the weight percent of PVA and SP ratio was calculated according to the following equation:
SP ¼
Mpowder VPVA
ð1Þ
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Fig. 2. XRD spectrum of the porous Nb-Ti-Ta alloy with 60% porosity.
where M is the mass of corresponding powder (g) and V is the volume of PVA solution (ml). The mixed powder slurries were mainatined under constant magnetic stirring for 1 h, and then the sponges were carefully impregnated in the slurries. Subsequently, the specimens were dried at 40 °C for 24 h and heated at 400 °C for 2 h under a vacuum to burn out the sponge scaffolds. Subsequently the sintering process was carried out in high vacuum (≤9 × 10−3 Pa) at 1700 °C for 2 h with the heating rate of 10 °C/min and then furnace-cooled to room temperature.
2.2. Characterization of porous Nb-Ti-Ta alloy scaffolds The phase composition and morphology analysis were examined by X-ray diffraction (XRD, D/Max-2550, Japan) and A NOVATM Nano 230 scanning electron microscope (SEM) with an energy dispersive X-ray (EDX) unit. The apparent porosities of the scaffolds were measured by both mass-volume method and Archimedes principle, which was described as elsewhere [4,22]. Compression tests were carried out with a crosshead speed of 1 mm/min at room temperature by using an Instron
Fig. 3. Relationship between pore size distribution and porosity for porous Nb-Ti-Ta alloys: (a) P = 50%, (b) P = 60%, (c) P = 70% and (d) P = 80%. (e) Relationship between average pore size and porosity for porous Nb-Ti-Ta alloys.
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phosphate. Alloys were sterilized with autoclave at 120 °C for 30 min, then incubated with 1 ml culture medium containing 5 × 104 cells in a humidified atmosphere with 5% CO2 for 4 h. All the samples with cells were transferred into 24-well plates so that the attachment could happen. After the desired time (6 h, 48 h and 7 days) culture, the media were removed and washed by phoaphate buffered saline (PBS), then pre-fixed in 3% glutaraldehyde for 30 min. Gradient ethanol dehydration and subsequent gradient hexamethl-disilazane dehydration were followed. Cellular attachment, spreading behavior and detailed morphologies of as-obtained specimens could be observed by SEM after Au sputtering. 2.4. In vivo test in a rabbit model
Fig. 4. The compressive strength and elastic modulus of porous Nb-Ti-Ta alloys with different porosities and pure Nb, Ta [24] made by the same process with 60% porosity. For comparison, the corresponding parameters of cancellous bone and cortical bone are listed from Ref. [24].
3369 maching. The slope of the initial linear portion of the stress-strain curve was used to define the elastic modulus and the compressive strength. At least five parallel tests were conducted for each group to ensure the reproducibility. 2.3. Apatite-forming ability and in vitro experiments 2.3.1. Apatite formation Apatite-forming test was carried out by immersing the samples into SBF solution (placed in 100 ml conical flask) at 37 °C for different time (3 days, 9 days and 14 days) and the SBF solution was refreshed every 2 days. After being immersed, samples were taken out and cleaned with distilled water, then dried in an oven at 40 °C for further characterization. 2.3.2. Cell adhesion and proliferation Human osteoblast-like MG-63 cells were seeded and cultured with medium consisting of RPMI 1640 supplement with 10% (v/v) fetal bovine serum (FBS, Thero), 1% antibiotics and 0.85 mM ascorbic acid-2
Fig. 5. SEM images showing the microstructure of sintering neck of porous Nb-Ti-Ta alloy.
The animal experimentation protocol was submitted and approved by local animal care and safety committee. The experiments were performed on adult New Zealand white rabbits weighting 2.5–3.0 kg. The rabbits were kept individually in stainless steel cages, which provided standard food and sterilized drinking water with unrestricted mobility and were checked daily. All animals were operated under general anesthesia. The rabbits were executed after 12 weeks, and the implant-bone compounds were harvested. All samples were dried in an oven and the detailed morphologies of as-obtained specimens could be observed by SEM after Au sputtering. 3. Results 3.1. Pore structure Porous Nb-Ti-Ta alloys with different porosities are shown a good 3D interconnected network and the anisotropic porous structure was very similar to that of trabecular bone, especially for the case with a higher porosity level of 60%–80%. As shown in Fig. 1, with the decrease of porosity, the interconnectivity became poorer. The pore connectivity was crucial for transformation of body fluids when the scaffolds were implanted into human body and it was also of great importance during surface modifications for chemical solutions throughout the pores. The pore connectivity was mainly achieved from the macro pores, which resulted from the macroporosity of sponge scaffolds and micro pores were formed by the evaporation of PVA and gradual elimination of the sponge scaffolds during the following sintering process. Previous investigations [1,4] have demonstrated that equiaxed pores were less efficient than the anisotropic porous structure, thus it indicate that the present porous Nb-Ti-Ta alloys possessed the anisometric pore structure, which was suitable for bone implants and could reduce “stress shielding” effect. Table 1 displays the porosities measured by Achimedes principle in association with the calculated values by mass-volume method for porous Nb-Ti-Ta alloys. It was obvious that the difference between the measured and calculated data was b3%, indicating that the above-mentioned process could excellently control the porosity. XRD spectrum (Fig. 2) of the porous alloys only consisted of β phase. No hydride, oxide or other impurities were detected in the XRD patterns. It indicates that the manufacturing process did not introduce any other impurities, which have detrimental effects on the mechanical properties and biocompatibility of the materials. Quantitatively, pore size distribution of the porous Nb-Ti-Ta alloys in terms of the mean diameter using image analysis method based on the SEM image was shown in Fig. 3, in which hundreds of pores were measured to confirm the average pore size. For the alloy scaffold with 50% porosity, pore size was relatively homogenous and N85% pore sizes were in the range of 50–300 μm and none of pores was larger than 400 μm. With the increase in porosity, the pore size distribution became wider and the maximum pore size became bigger. As the porosity level reached to 70%, almost 90% pores were in the range of 150–400 μm, as shown in Fig. 3(a)-(d). Furthermore, Fig. 3(e) exhibited the relationship between pore size and porosity for the porous alloy scaffolds. The
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Fig. 6. SEM images showing the microstructure of porous Nb-Ti-Ta alloys after immersion in SBF for (a) 3 days, (b) 9 days and (c) 14 days.
average pore size of porous alloy scaffold with 50% porosity was 199 μm, and the average pore size increased to 317 μm when porosity reached to 80%. Qualitatively, porosity, pore size and its distribution of porous NbTi-Ta alloy scaffolds could be tailored in the present fabrication process. As the pore size considerably influenced the biocompatibility of porous implants [7] and pore size in the range of 100–600 μm was well suited for the ingrowth of new bone tissues [1,7], the present porous Nb-TiTa alloy scaffolds could have the capability of matching different requirements of the human bones. 3.2. Mechanical properties In the present work, the relationship between the elastic modulus and the compressive strength is shown in Fig. 4. Both the modulus and the strength decreased with the increase of porosity. The compressive strength and Young's modulus of the porous Nb-Ti-Ta alloy with different porosities, achieved in the range of from 17.45 ± 2.76 MPa to 121.67 ± 1.76 MPa and 0.11 ± 0.01 GPa to 2.08 ± 0.09 GPa. Young's modulus and compressive strength of human trabecular bones are in the range of 0.1–30 GPa and 2–70 MPa with different values depending on age and experimental conditions [23]. Metallic biomaterials for medical applications are suffering from the “stress shielding” effect, which could lead to bone resorption and implant loosening. Therefore, the elastic modulus and compressive strength of medical implants should be well tailored to match different requirements of the human bones and controlled in the range of 0.1–30 GPa and around 100 MPa, respectively [24]. It should be noted that the developed porous Nb-Ti-Ta alloys processed the optimum compressive strength around 100 MPa, which was in the range of between cancellous bone and cortical bone, and could meet the mechanical properties of trabecular bone well. Moreover, the
Young's modulus of the porous scaffolds was falling in the range of between cancellous bone and cortical bone, which had the potential to minimize the stress shielding effect. As well known, it is hard to obtain a good balance between suitable modulus and enough compressive strength for porous scaffolds. The SEM micrograph (Fig. 5) shows the morphology of sintering neck between particles formed during diffusion bonding, which would strengthen the bond between particles and lead to the shrinkage of the micropores, thus offering a balance between the high porosity and enough load bearing ability. It is also obvious that there are no unmolten powders and impurities on the surface of porous structure, which may present in the samples fabricated by a rapid prototyping and a space-holder method and could show detrimental effects on the mechanical properties [7]. 3.3. In vitro studies with SBF and cell adhesion Stimulated body fluid (SBF) was used to evaluate the apatiteforming ability of the obtained alloys, and the materials can achieve the bone-bonding process via the induced apatite formation in body fluids [25]. Typically, Fig. 6 compares the morphologies Nb-Ti-Ta alloy scaffolds after immersion in SBF for 3 days, 9 days and 14 days. It was found that all the surfaces induced nucleation of particles after immersion in SBF, the density of the particles in the scaffolds increased with immersing time. Fig. 6 (c) presents that the precipitated particles became coarser and homogenously with dome shaped particles after 14 days immersion in SBF. It should be noted that the rough porous surface may act as better sites for the nucleation and growth of particles, which benefits the formation of apatite. Furthermore, to evaluate the in vitro biocompatibility, the adhesion and proliferation of MG-63 cells on the porous Nb-Ti-Ta alloy scaffolds is shown in Fig.7(a)(b). It can be observed that the elongated and
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Fig. 7. SEM micrographs showing the surface morphologies of Nb-Ti-Ta alloys after MG-63 cell cultured for (a) 6 h, (b) 48 h and (c) 7 days.
flattened MG-63 cells have formed numerous filopodia and lamellipodia, anchoring on the surface and into the internal surface of the pores. Qualitatively, after 7 days culture (Fig. 7(c)), cells could spread well and established connection with neighbor cells, suggesting that the porous alloy scaffolds could offer a good/excellent adhesion and proliferation ability. 3.4. In vivo test The SEM images showed the microstructure of porous Nb-Ti-Ta alloys after implanted on rabbit model for 12 weeks, as shown in Fig. 8. After 12 weeks implantation, the original porous scaffolds were covered with newly born bone tissues, which showed no obvious gap. From the high magnification image on an area of interest, the bone-trabecular like areas confirmed the new bone tissue generation, as shown in Fig. 8(b). Furthermore, the Ca/P ratio of the formed bone tisssue were detected by EDS, and the result fixed at 1.25, which was lower than that of natural HA of 1.67. 4. Discussion 4.1. Structure of porous alloy scaffolds Up to now, various fabrication processes have been proposed to prepare porous alloys with elongated pores, which only possess equiaxed pores [2]. Porous alloys with equiaxed pores are often manufactured by simply controlling the aspect ratio and the amount of pore-foaming agent which is difficult in reducing “stress shielding” effect and could hardly adapt to the complex human body environment. For porous materials with potential for trabecular bone implant applications, the structure of pores is a key factor that can affect long term
fixation. In order to further reveal the porous structure of Nb-Ti-Ta alloys fabricated by the combination of the sponge impregnation technique and sintering method, Fig. 9 presents schematic diagrams of the fabrication methods for some widely used metallic implants. From Fig. 9(a), for porous titanium fabricated by diffusion bonding of titanium meshes, the porosity and pore size could be controlled by adjusting the aspect ratio of titanium meshes. Therefore, the pore was regular and exhibited a latitude-longitude like structure, which could be difficult to adapt to the complex structure of human bones. From Fig. 9(b), porous Ti-18 at.% Nb-4 at.% Sn alloy was prepared by a space-holder method using NH4HCO3 as space-holder. However, it is difficult to possess porous alloys with excellent design and accurate control of the porosity by simply adjusting the amount of space-holder in abovementioned method [26,27]. For the present fabrication method of porous alloy scaffolds, the sponge scaffolds were impregnated into the mixed powder slurries during the process of impregnation. Simultaneously, the skin of the pores delivered the mixed powder slurries to different inter-connected surface of pores due to the drive of both extrusion force and capillary force. Subsequently, in the following dry process, the water in slurry was evaporated; the powder particles were bonded together on the internal surface of the pores of the sponge scaffolds. Finally, in the sintering process with high vacuum, the pores size came to shrink through gradual elimination of the sponge scaffolds and thus achieved the formation of the 3D porous structure with elongated pores. As apparently seen in Fig. 10, the present porous Nb-Ti-Ta alloy scaffolds could attract much more attention due to their morphological similarity to the anisotropic porous structure of bones. B. Vafaeian et al. [28] simulated the porous trabecular bone-mimicking structure based on the micro-scale finite element method (FEM) and both the numerical simulation and experimental results were well agreed with Fig. 10.
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Fig. 8. (a) SEM images showing the morphologies of porous Nb-Ti-Ta alloys after in vivo test with rabbit model for 12 weeks; (b) high magnification of newly born bone tissues and (c) EDS result of newly born bone tissues.
4.2. Effect of porosity on compressive properties Theoretically, the compressive strength and elastic modulus of a porous solid are closely related to the porosity level, distribution and sizes. To design porous scaffolds with proper mechanical properties, the
relationship between compressive properties and porosity should be studied quantitatively. Gibson et al. [14] have demonstrated in detail that compressive properties of porous solids with different porosities, and established equations well known as Gibson-Ashby model to illustrate the relationship. According to Gibson-Ashby model, the
Fig. 9. Outline of fabrication method of porous alloys: (a) diffusion bonding of titanium meshes [21] and (b) space-holder method process [32].
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Fig. 10. (a) Schematic diagrams showing the process of poring by sponge impregnation technique; (b) SEM micrograph showing the morphology of cancellous bone.
relationship between the compressive strength, elastic modulus and porosity can be expressed as follows: E=Es ¼ C1 ðρ=ρs Þn1
ð2Þ
σ pl =σ ys ¼ C2 ðρ=ρs Þn2
ð3Þ
where E, σpl and (ρ/ρs) are Young's modulus, plateau stress and relative density of porous solids, respectively. Es and σys are elastic modulus and yield strength of corresponding metals, respectively. C1 and C2 are constants depending on the porous structure [14]. A regression analysis of the mechanical properties of the porous NbTi-Ta alloys was performed, as shown in Fig. 11. The exponents n1 and n2 were determined to be around 2.0 and 1.3, respectively. It indicated that the relationship between the mechanical properties and the relative densities of the porous alloy scaffolds could conform to the Gibson-Ashby model when C1 = 0.08 and C2 = 0.61. Therefore, the compressive properties of the porous scaffolds fabricated by the present method could be well predicted and the desired mechanical properties could be tailored by porosity to match those of the human bones based on the Gibson-Ashby model.
4.3. Effect of pore on biocompatibility behavior Structurally, several key aspects play important roles for successful porous scaffolds as medical applications. Previous studies [4,29,30] have pointed out that parameters, such as porosity level, pore sizes and morphology, had direct effects on the interaction between implants and their surrounding tissues. Moreover, a large number of research papers have identified that the macropores in the range of 100–600 μm can evidently promote bone tissue ingrowth and eventually achieve good implant fixation [4]. The micropores (5–20 μm) could offer a considerably good fixation of earlier cell attachment due to a bigger surface area and roughness of pore surfaces [4,30–32]. Another important parameter is open porosity. As well known, the better connectivity of porous materials is, the more connected channel for cell ingrowth. Because it is helpful for cells to adhere, proliferate and differentiate as well as offer channels to allow nutrient delivery and waste removal. In the present work, the prepared porous Nb-Ti-Ta alloys showed a 3D interconnected porous structure with the open porosity as high as 90%. The spontaneously formed oxides will provide a bio-inert layer on the alloy surface in the complex human body fluids [4,33]. Therefore, the enrichment of oxides on the surface can suppress the dissolution
Fig. 11. Relationship between compressive properties and relative density for porous Nb-Ti-Ta alloys.
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Fig. 12. Schematic diagrams showing the mechanism of apatite formation on Nb-Ti-Ta alloy in SBF.
of Nb, Ta and Ti ions, which may play a significant role in determining the interaction between implant materials and surrounding tissues. Furthermore, previous studies showed that Ti-OH can be formed in Tibased alloys after SBF immersion [34,35]. As shown in Fig. 12, the mechanism of apatite formation on the Nb-Ti-Ta alloys in SBF was interpreted in terms of electrostatic interactions between the surface and the ions in SBF. Ti-OH as well as Nb-OH [35,36] and Ta-OH [35,37] could be formed on the surface of Nb-Ti-Ta alloys, which is beneficial to the formation of apatite for a long term. Previous studies confirmed that the morphology of cells grown on the surface of alloys could reveal the interactions between the cells and the surface of scaffolds, especially in the early phases of culture [38–40]. As shown in cell attachment and adhesion morphology from the SEM micrographs, MG-63 cells cultured on porous Nb-Ti-Ta alloy scaffolds preferentially presented both lamellipodia and filopodia, anchored on the surfaces and even into the internal surface of the pores. According to the previous results obtained with the cells on dense alloys [41–43], these morphologies reveal more tightly adhering cells. It is known that the in vitro biological test could reflect the early in vivo response, so we can hypothesize that porous Nb-Ti-Ta alloy scaffolds could enhance cellular proliferation by stimulating the transforming growth factor-β (TGF-β) and induce early differentiation via promoting the transcription of alkaline phosphatase (ALP) [44].
5. Conclusion (1) Highly porous Nb-Ti-Ta (at.%) alloys with pore sizes of 100–600 μm and porosity level of 50%–80% were fabricated by the combination of the sponge impregnation technique and sintering method, which were capable of making 3D interconnected anisotropic porous structure. The porous scaffolds provided a compressive Young's modulus of 0.11 ± 0.01 GPa to 2.08 ± 0.09 GPa and the strength of 17.45 ± 2.76 MPa to 121.67 ± 1.76 MPa. (2) The relationship between compressive properties and porosity levels was well consistent with the Gibson-Ashby model and the mechanical properties could be tailored to match different requirements of the human bones. (3) The SBF test revealed that porous Nb-Ti-Ta alloys had the potential to induce apatite in vitro, which was evidently beneficial to the interaction between implant materials and surrounding tissues in human body fluid environment. Porous Nb-Ti-Ta alloys also had an excellent biocompatibility, which could enhance adhesion of MG-63 cells. Moreover, the porous structure could induce cells into the pores, offering good biological fixation and the bone-trabecular like areas from the in vivo test further confirmed the new bone tissue generation.
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