J Mater Sci: Mater Med (2016) 27:76 DOI 10.1007/s10856-016-5685-6
BIOMATERIALS SYNTHESIS AND CHARACTERIZATION
Original Research
Preparation and characterization of biomedical highly porous Ti–Nb alloy Jianming Ruan1 • Hailin Yang1 • Xiaojun Weng2 • Jinglei Miao3 • Kechao Zhou1
Received: 10 October 2015 / Accepted: 29 January 2016 Ó Springer Science+Business Media New York 2016
Abstract The compressive strength and the biocompatibility were assessed for the porous Ti–25 wt%Nb alloy fabricated by the combination of the sponge impregnation technique and sintering technique. The alloy provided pore sizes of 300–600 lm, porosity levels of 71 ± 1.5 %, in which the volume fraction of open pores was 94 ± 1.3 %. The measurements also showed that the alloy had the compressive Young’s modulus of 2.23 ± 0.5 GPa and the strength of 98.4 ± 4.5 MPa, indicating that the mechanical properties of the alloy are similar to those of human bone. The scanning electron microscopy (SEM) observations revealed that the pores were well connected to form threedimension (3D) network open cell structure. Moreover, no obvious impurities were detected in the porous structure. The experiments also confirmed that rabbit bone mesebchymal stem cells (MSCs) could adhere and proliferate in the porous Ti–25 wt%Nb alloy. The interactions between the porous alloy and the cells are attributed to the porous structure with relatively higher surface. The suitable mechanical and biocompatible properties confirmed that this material has a promising potential in the application for tissue engineering.
& Hailin Yang [email protected] 1
State Key Laboratory of Powder Metallurgy, Central South University, Changsha 410083, People’s Republic of China
2
Department of Joint Surgery & Sports Medicine, Orthopaedic Centre, Hunan People’s Hospital, Changsha 410002, People’s Republic of China
3
Department of Orthopedics, Third Xiangya Hospital of Central South University, Changsha 400013, People’s Republic of China
1 Introduction Of all metallic biomaterials, Ti-based alloys have several favourable advantages in corrosion resistance, biocompatibility, low elastic modulus, density and the capacity of easily binding with human bone and other types of tissue. Although the metallic ions such as V, Co, Al, and Ni released from Ti-based alloys can generate detrimental biological effects in the complex human body fluid environment [1–3], the elements including Ti, Nb, Ta and Zr are found as non-toxic and capable of providing good biocompatibility [4–6]. Therefore, Ti-based alloys containing Ti, Nb, Ta and Zr are widely used in manufacturing biomedical implants [7, 8]. Moreover, the Ti, Nb and Ta oxides generated in the complex human body environment are also identified to have good biocompatibility, good corrosion resistance and non-toxic [4]. This further promotes the applications of Ti-based alloys as bio-materials. One of the challenges impeding Ti-alloys wide application is their high modulus in comparison with that of human bones. To solve the problems, porous materials are becoming favourable for the clinical application of structural bone graft and bone regeneration in recent years [9, 10]. Because the size and distribution of the porosity in porous materials can be controlled, therefore, the structure modulus can be adjusted to meet the specific requirements. Moreover, the porous structure can provide a good biological fixation to the surrounding tissue through inducing bone tissue growing into the porous cell [11]. Several methods [12–15] have been reported to fabricate porous metallic biomaterials, which typically include selfpropagating high-temperature synthesis (SHS), hot isostatic pressing (HIP), space-holder sintering process (SSP) and other conventional sintering (CS). Aguilar Maya et al. [16] fabricated a porous Ti–10Zr–10Nb alloy by using space-
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holder sintering to reach a porosity level of 70 % and a porous size of 260 lm. The material exhibits a Young’s modulus of 3.9 GPa and a compressive plateau stress of 67 MPa. Sirikul et al. [17] also synthesized a porous NiTi alloy by SHS technique, which have a porosity of 42–58 % and pore sizes between 340 and 500 lm, and the open porosity was controlled in the range of 87–98 %. However, the currently available methods are still difficult to construct the good porous alloys for tissue ingrowth through controlling the pore sizes, pore structure and mechanical properties. In the present work, we introduce a porous Ti– Nb alloy fabricated by a method of integrating of the sponge impregnation and sintering technique. The characteristics of the porous Ti-Nb alloy and the mechanical properties were evaluated and the biological behaviour of the porous Ti–Nb alloy was assessed.
2 Experimental 2.1 Preparation and characterization of porous Ti– Nb alloy scaffolds
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The apparent porosity and the open porosity of the Ti– 25 wt%Nb alloy were measured by the Archimedes method, which is described in detail elsewhere [18, 19]. Compression tests for mechanical property evaluation were performed by using an Instron3369 Universal Electromechanical Testing System equipped with Bluehill software with 250 KN capacity at a constant crosshead speed of 0.5 mm/min. Young’s modulus and compressive strength were determined from the stress–strain plots derived from load–displacement data recorded during compression testing. The reported data were obtained from averaging the results of five samples. All the tests were performed at ambient temperature (*25 °C). The phase composition and crystallinity of Ti– 25 wt%Nb alloy samples were characterized by X-ray diffraction (XRD) analysis at room temperature. A Jeol6360LV scanning electron microscopy (SEM), equipped with an EDAX-Phoenix energy dispersive spectroscopy X-ray (EDX) analysis system to analyse elemental distribution of the porous Ti–25 wt%Nb alloy, was utilized to analysis cross sectional microstructures. 2.2 Isolation and culture of MSCs
The starting elemental metal powders of TiH2 (with a purity of 99.9 % and an average diameter of 45 lm), Niobiumhydrides powder (with a purity of 99.5 % and an average diameter of 9.97 lm) were weighed to give a nominal composition of Ti–25 wt%Nb. These powders were blended together by ball milling for 2 h in planetary ball mill at the rate of 200 rpm. A commercial polyurethane sponge (Dongguan Zhanyu Industry & Trade Co., Ltd., P. R. China) employed in the experiment has an interconnected macroporosity, with an open porosity of 50–80 pores per inch (PPI), which corresponds to pore diameters 500–850 lm. And the density of the sponge is in the range of between 0.05 and 0.07 g/cm3. Before impregnating mixed powder slurry into the sponge, the sponge was cut in blocks of A10 9 25 mm3 and was repeatedly washed in 10 % NaOH solution at the temperature of 40–60 °C for 30 min, then swilled with deionized pure water for six times. In order to control the slurry viscosity and optimize the ability of blended powders to coat the sponge, 5 % (wt%) polyving akohol(PVA)aqueous solution was used as a binder. The slurries were kept under constant magnetic stirring during 1 h, and then the specimens were carefully impregnated in the slurries. Subsequently, the specimens were dried in vacuum at 100 °C for 24 h. Finally, the sponge scaffolds and the PVA were burned out from the green body at 300 °C for 3 h and the subsequent sintering process was carried out in a high vacuum furnace (below 9.0 9 10-4 Pa) at 1350 °C for 2 h with the heating/cooling rate of 10 °C/min.
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Rabbit bone marrow mesenchymal stem cells (MSCs) were derived from healthy 2.4 kg 8-week old male New Zealand white rabbit. Rabbit bone marrow-derived MSCs were flushed out via aspiration from the femoral bone marrow iliac crest by using a 16-gauge syringe needle, collecting 3 ml of marrow blood into 100 u/ml of heparin (2 ml). This cell suspension was placed over a Ficoll solution (1.073 g/ ml) slowly. The marrow blood was filtered through a cell strainer to exclude any fatty tissues and blood clots, and processed in a centrifuge at 2000rmp for 15 min. Subsequently, nucleated cells at the medium-Percoll interface were collected, then washed with 10 % PBS medium (including penicillin 100 l/ml and streptomycin 100 lg/ml) and plated in culture flasks at 1.0 9 106/cm2 with complete medium. Culture flasks were maintained in a humidified inculcator at 37 °C with 5 % CO2. Adhered cells were fed by complete replacement of the medium per 3 days. When the layer was 80 % confluent, the culture was trypsinized and cells were subcultured onto two or three culture flasks at a cell density of 1 9 104 cells/cm2. Cells underwent three passages in culture to ensure that all contaminating hematopoitic cells had been totally washed out. Detached cells were washed and re-suspended in PBS, and incubated with the following mono clonal antibodies directly conjugated with fluorescein isothiocyanate (FITC): CD29, CD34, and CD44. Cells were then washed and immunophenotypic analysis was then performed by flow cytometry.
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2.3 Seeding of MSCs on porous Ti-Nb alloy surface
2.5 Statistical analysis
The specimens were cut into U10 mm 9 2 mm with electrical discharge wire cutting and gently polished with 1000# sand paper. Then, they were sterilized in a highpressure autoclave at 120 °C for 30 min. Following this, MSCS of the 3rd passage were then seeded onto sample surfaces and placed in a 12-well plate. The initial cell density was 1 9 105 cell per wall. 2 milliliters of Dulbeccos modified Eagles medium (DMEM) enriched with 10 % fetal bovine serum was added to each well. MSCs were stained with acridine orange after 3, 24 and 72 h of incubation to assess the adhesion and proliferation by fluorescence microscopy. And the cell-material complex of 72 h of incubation was isolated, fixed with 3 % glutaraldehyde for 30 min, dehydrated with ethanol and dried in vacuum. Dried samples were then gold-coated and observed under SEM.
All the data were analysed using SPSS (version 15.0) software for windows student version. The differences among the groups were analysed through one-way analysis of variance (ANOVA). A p value of less than 0.05 was considered to be statistically significant..
2.4 MTT assay 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was utilized to determine the viable cell numbers based on the reductive cleavage of MTT (a yellow salt) to formazan (a dark blue compound) by mitochondrial dehydrogenase of living cells. Cells were seeded in 96-well plates with cell seeding density of 1 9 105 in a humidified incubator containing 5 % CO2 at 37 °C for 24 h. The wells were divided into three groups for the following tests: (1) the porous Ti–25 wt%Nb alloy group, the cells were cultured in the extract of the porous Ti–25 wt%Nb alloy (extracted 1 g material with 5 mL medium); (2) the bulk Ti–25 wt%Nb alloy group, the cells were cultured in the extract of the porous Ti–25 wt%Nb alloy (extracted 1 g material with 5 mL medium); (3) negative control group, the cells were cultured in fresh RPMI-1640 medium. Then, after being incubated for 1, 2, 3 and 7 days, cells were incubated for another 4 h with 10 lmol/L of MTT (Thiazolyl Blue Tetrazolium Bromide; Gibco,USA), respectively. Washing with PBS was followed by the addition of DMSO(dimethyl sulfoxide; Gibco,USA) (150 lL) and gently shaking for 10– 15 min to ensure the complete dissolution. The optical density (OD) of the solution in each well was measured after 60 min of slow shaking the absorbance at a wavelength of 492 nm using a plate spectrophotometer. The relative growth rate (RGR) was calculated according to the following equation: RGR ¼ ODsamples =ODnegative control 100%
ð1Þ
The criteria for cell toxicity classification were defined as: RGR C 100 %, Level 0; RGR 75–99 %, Level I; RGR 50–74 %, Level II; RGR 25–49 %, Level III; RGR 1–24 %, Level IV; RGR 0, Level V.
3 Results 3.1 Material characterization The microstructure of porous Ti–25 wt%Nb alloy is shown in Fig. 1. It can be seen from Fig. 1a that pores on the surface of Ti–25 wt%Nb alloy showed a good 3D interconnected network with the pore size of between 300 and 600 lm, which was smaller than the size of sponge scaffolds (500–850 lm). Obviously, this is because the formation and the growth of sintering neck between powder particles make the origin pores shrink. During the process of impregnation, the sponge scaffold was impregnated into the blended suspension liquid. Simultaneously, in the sponge scaffold the skin of pores delivered the suspension liquid to different inter-connected surface of pores due to the drive of capillary force. And, subsequently, in the following high vacuum dry process, the water in PVA was evaporated; the powder particles were bonded together and stuck on the skin of pores. In the final sintering process the pore sizes came to shrink through gradual elimination of the sponge scaffold and mechanical bonding of solid particles. Figure 1b presents the morphology of human cancellous bone. It can be found that the porous structure of prepared highly porous Ti–25 wt%Nb alloy was similar to human cancellous bone. Moreover, it should be noted from Fig. 3a that there were many second pores distributed on the wall of the pores and their sizes of the pores are in the range of 5–20 lm, which could be attributed to the shrinkage of sintered particles. It was determined by Archimedes method that the porous Ti–25 wt%Nb alloy possessed a porosity of 71 ± 1.5 %. The porosity was a slight lower than that of the sponge scaffold. Moreover it is worth being pointed that the open porosity could achieve as high as 94 ± 1.3 %. The good interconnected network of sponge scaffold and considerable large pores (as high as 500-850 lm) ensured the carriers with powders could go along with skin of sponge pores rather than block them. XRD spectrum (Fig. 2) of the Ti–25 wt%Nb alloy, as expected, revealed that a ? b alloy and no other obvious impurities peak occurred. This indicates that during the sintering process the high vacuum (as low as 10-4 Pa) would ensure that the scaffold and the binder could be
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(a)
(b)
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Fig. 1 a SEM micrograph showing the morphology of pores in the Ti–25 wt%Nb alloy; b SEM micrograph showing the cancellous bone of human bone
(a) (a) Intensity/ a.u
--- α --- β
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Ti La
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Element Nb L Ti K Matrix
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Fig. 2 XRD spectrum of the porous Ti–25 wt%Nb alloy
76.95
At% 13.38 86.62
Correction
ZAF
Wt% 23.05
1.9
burned out or volatilized effectively. Furthermore, the composition was confirmed by EDS, as shown in Fig. 3b, that Nb concentration was 23.05 wt%, approaching to the designed concentration of 25 wt%.
1.3 0.6 0.0
3.2 Mechanical properties Metallic biomaterials currently in use for medical applications are suffering from their higher elastic modulus than human bone (0.1–30 GPa) and lacking of sufficient osseointegration for implant longevity [20]. If the elastic modulus of those biomaterials is much greater than that of human bone, the bone-producing cells called osteoblasts are stimulated into generating more bone when the bone is stressed [21]. This will thereby further result in an occurrence of bone resorption, increasing the healing time and even finally implant surgery failure [22]. Therefore, the elastic modulus and the compressive strength of these biomaterials should be controlled in the range of human bone from 0.5 to 30 GPa and around 100 MPa, respectively [19]. In present work, the compressive strength and Young’s modulus of the porous Ti–25 wt%Nb alloy,
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Nb La
Ti La
Ti La
1.00
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4.00
5.00
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Fig. 3 a SEM micrograph showing the detailed morphology of porous wall surface of the porous Ti–25 wt%Nb alloy and b the EDS diagram and the analytic composition of metallic skeleton (marked by red square on SEM micrograph) of the Ti-25 wt%Nb alloy (Color figure online)
measured from the compression mechanical test, achieved 98.4 ± 4.5 MPa and 2.23 ± 0.5 GPa, respectively. 3.3 Cell culture and proliferation Figure 4 shows immunophenotypic analysis by flow cytometry to MSCS of the 3rd passage. It can be seen that CD44 were positively expressed in 96 %, CD29 in 95 %, but only CD34 in 5 % MSCs, which indicates that the 3rd passage of MSCs maintained standard immunophenotypy of MSCs.
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Fig. 4 Immunophenotypic analysis by flow cytome try to MSCS of the 3rd passage
Qualitatively, florescent optical micrographs of the adhesion and proliferation of MSCs on the porous Ti– 25 wt%Nb alloy are presented in Fig. 5. For better comparison, the bulk Ti–25 wt%Nb alloy was selected to assess its biocompatibility together with the porous Ti– 25 wt%Nb alloy. As seen, with increasing inoculation time, the adhesion and proliferation of MSCs on both materials showed a significant increase. Moreover, compared with the bulk Ti–25 wt%Nb alloy, the porous Ti– 25 wt%Nb alloy showed evidently much more MSCs on its surface. Furthermore, quantitatively, the total numbers of MSCs of the adhesion and proliferation on the both materials counted by fluorescence microscope is showed in Fig. 6. As expected, the adhesion and proliferation of MSCs on the porous Ti–25 wt%Nb alloy showed much more rapidly than that of the bulk Ti–25 wt%Nb alloy with increasing period of inoculation time from 3 to 72 h (P \ 0.05). Typically, the SEM morphology of MSCs on the porous Ti–25 wt%Nb alloy surface after 72 h is shown in Fig. 7. It can be seen that MSCs showed flatter or irregular morphology with the size of 5–20 lm, which partially covered
on the surface of porous Ti–25 wt%Nb alloy scaffold or extended inward the pores. The cells clustered and were abounded with pseudopodium, showing better adhesion and activity.
3.4 Cytotoxicity results In present study, the MTT assay was utilized to quantitatively determine the proliferation of the viable MSCs on both of the porous Ti–25 wt%Nb alloy and the bulk Ti– 25 wt%Nb alloy. Figure 8 shows a comparison of viable cell densities for the porous Ti–25 wt%Nb alloy, the bulk Ti–25 wt%Nb alloy and negative group after different cultured time of 2, 4 and 7 days. As seen, the optical density (OD) of the porous Ti–25 wt%Nb alloy is significantly higher than that of the bulk Ti–25 wt%Nb alloy and is just slightly lower than that of the negative group. Furthermore, the results of toxicity of both materials by MTT assay indicated that the RGR values of cells were in the range of between 94 and 113 %, indicating that porous materials were insignificant cytotoxicity (Level I).
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Fig. 5 Florescent optical micrographs showing the morphology of MSCs on the surface of bulk (a–c) and porous (d–f)- Ti–25 wt%Nb alloy cultured at different times, a and d 3 h, d and e 24 h, c and f 72 h 300
Bulk Ti-25 wt.% Nb alloy
Cell Number
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Porous Ti-25 wt.% Nb alloy
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Fig. 6 Cell numbers of the MSCs adhering on the bulk and porous Ti–25 wt%Nb alloy at different times
20μm
Fig. 7 SEM micrograph showing the surface of the porous Ti– 25 wt%Nb alloy after culturing 72 h in ambient environment (25 °C)
4 Discussion 4.1 Biomechanical behaviour Just mentioned earlier, suitable mechanical properties, such as modulus and strength, are quite important. The modulus of the bulk bio-metallic materials is more than 40 GPa according to the recent reports [23–26]. The effective way to low down the modulus is applying porous design to the metal. But, on the other hand, the decreasing modulus always results in a rapid decrease of strength. Therefore, getting the balance of suitable modulus and compressive
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strength for the porous alloy is critical. Reviewing our previous results [18, 19], as presented in Fig. 9, it can be found that the present porous Ti–25 wt%Nb alloy have the optimum compressive strength, which is higher than that of the porous Ti-50Ni alloy [19] and the porous Ta [18]. And the compressive strength is in the range of between cancellous bone and corticall bone [19], and met the mechanical properties of requirement of trabecular bone well. On the other hand, the elastic modulus of the porous Ti–25 wt%Nb alloy, similar to the porous Ti–50Ni alloy and the porous Ta are falling into the range of between
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Optical density
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Bulk Ti-25 wt.% Nb alloy Porous T- 25 wt.% Nb alloy Negative group
0.9 0.6 0.3 0.0
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Fig. 8 MTT assay of the bulk and porous Ti–25 wt%Nb alloys cultured at different times
Compressive strength /MPa
200 4.4~28.8 GPa
160 120
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0.50 GPa 3.99
0.07 GPa 2.21
0.16 GPa
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0.01~3.0 GPa
40 0
Ti-25Nb
Ti-50Ni
Ta
Cancellous bone
Corticalbone
Fig. 9 The compressive strength of the different alloys made by the same process with the same volume fraction of pores at a level of 70 %
cancellous bone and corticall bone. This fact is very attractive for its potential to minimize the stress shielding effect and to optimize short term and long term fixation, indicating that the porous Ti–25 wt%Nb alloy have a good biomechanical compatibility. 4.2 Biocompatibility behaviour Structurally, for a successful biometal porous implant, many aspects take important roles. First of all, the pore parameters should have specific range for in-growth of human bone tissues. Many previous studies [22, 27] have pointed out that many parameters, such as porosity, porous size and morphology, have great effects on the ability of interaction with cells. Porous Ti alloys are expected to offer better surface interaction area with cells by offering mechanical anchoring sites, in turn, facilitating the growth of cells [23]. Furthermore, an increasing number of investigations identified that the pore size of 100–600 lm can more easily enhance the bone tissue ingrowth and eventually good implant fixation. Our previous results [18,
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19] also revealed that the size of 300–600 lm of Ta and TiNi alloy can evidently promote the cells to ingrowth. Another important pore parameter is open porosity. As we know, the connectivity of porous material is extremely helpful. Because there are much more connected channel for adhesion, proliferation and differentiation when cells are attached to the material surface, as well as offering more connected channels to allow nutrient delivery, and waste removal. In the present study, the prepared porous Ti–25 wt%Nb alloy showed good pore structure mimicking human bone with 3D interconnected porous network of microscale (Fig. 1b), which can meet the requires mentioned above. As well as our previous investigations, the second pores in the range of 5–20 lm can offer a considerably good fixation of earlier cell attachment due to bigger surface area and roughness of pore surfaces, which is in according to some other findings [7, 28, 29]. Compositionally, the chemical components of the implant alloy play a significant role in determining the interaction between implant materials and their surrounding tissues. For a long term, spontaneously formed oxides whose compositions are Ti2O and Nb2O5, will provide a bio-inert layer on the alloys surface in the complex human body fluids [4]. Thereby, the enrichment of TiO2 and Nb2O5 on the surface suppressed the dissolution of Ti and Nb as ions. So, the Ti–25 wt%Nb alloy show high cell viability during the MTT assays.
5 Conclusions A high porosity of 71 %, with a 3D interconnected open porosity of 94 %, Ti–25 wt%Nb alloy was successfully prepared in combination of the sponge impregnation technique and sintering technique. This material can be capable of having a pore size of 300–600 lm, a compressive strength of 98.4 ± 4.5 MPa and compressive Young’s modulus of 2.23 ± 0.5 GPa. The Ti–25 wt%Nb alloy processes a ? b alloy, whilst the elements of Ti and Nb distributed uniformly and no obvious occurs during the preparation. The in vitro results revealed the highly porous Ti– 25 wt%Nb alloy have good biocompatibility, which can evidently promote adhesion, proliferation of MSCs. It is indicated that this porous Ti–25 wt%Nb alloy can induce steoblasts to penetrate into through the pores and interconnected channels, offering good biological fixation. All these data provide proofs further that porous Ti–25 wt%Nb alloy could be used as a good biomaterial in tissue engineering. Acknowledgments This work was supported by National Natural Science Foundation of China (No. 51404302,51274247), National
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High Technology Research and Development Program of China (863 Program) (No. 2012BAE06B00). The author Hailin Yang also would like to acknowledge the financial support from the independent project of State Key Laboratory of Powder Metallurgy Research Institute.
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BIOMATERIALS SYNTHESIS AND CHARACTERIZATION
Original Research
Preparation and characterization of biomedical highly porous Ti–Nb alloy Jianming Ruan1 • Hailin Yang1 • Xiaojun Weng2 • Jinglei Miao3 • Kechao Zhou1
Received: 10 October 2015 / Accepted: 29 January 2016 Ó Springer Science+Business Media New York 2016
Abstract The compressive strength and the biocompatibility were assessed for the porous Ti–25 wt%Nb alloy fabricated by the combination of the sponge impregnation technique and sintering technique. The alloy provided pore sizes of 300–600 lm, porosity levels of 71 ± 1.5 %, in which the volume fraction of open pores was 94 ± 1.3 %. The measurements also showed that the alloy had the compressive Young’s modulus of 2.23 ± 0.5 GPa and the strength of 98.4 ± 4.5 MPa, indicating that the mechanical properties of the alloy are similar to those of human bone. The scanning electron microscopy (SEM) observations revealed that the pores were well connected to form threedimension (3D) network open cell structure. Moreover, no obvious impurities were detected in the porous structure. The experiments also confirmed that rabbit bone mesebchymal stem cells (MSCs) could adhere and proliferate in the porous Ti–25 wt%Nb alloy. The interactions between the porous alloy and the cells are attributed to the porous structure with relatively higher surface. The suitable mechanical and biocompatible properties confirmed that this material has a promising potential in the application for tissue engineering.
& Hailin Yang [email protected] 1
State Key Laboratory of Powder Metallurgy, Central South University, Changsha 410083, People’s Republic of China
2
Department of Joint Surgery & Sports Medicine, Orthopaedic Centre, Hunan People’s Hospital, Changsha 410002, People’s Republic of China
3
Department of Orthopedics, Third Xiangya Hospital of Central South University, Changsha 400013, People’s Republic of China
1 Introduction Of all metallic biomaterials, Ti-based alloys have several favourable advantages in corrosion resistance, biocompatibility, low elastic modulus, density and the capacity of easily binding with human bone and other types of tissue. Although the metallic ions such as V, Co, Al, and Ni released from Ti-based alloys can generate detrimental biological effects in the complex human body fluid environment [1–3], the elements including Ti, Nb, Ta and Zr are found as non-toxic and capable of providing good biocompatibility [4–6]. Therefore, Ti-based alloys containing Ti, Nb, Ta and Zr are widely used in manufacturing biomedical implants [7, 8]. Moreover, the Ti, Nb and Ta oxides generated in the complex human body environment are also identified to have good biocompatibility, good corrosion resistance and non-toxic [4]. This further promotes the applications of Ti-based alloys as bio-materials. One of the challenges impeding Ti-alloys wide application is their high modulus in comparison with that of human bones. To solve the problems, porous materials are becoming favourable for the clinical application of structural bone graft and bone regeneration in recent years [9, 10]. Because the size and distribution of the porosity in porous materials can be controlled, therefore, the structure modulus can be adjusted to meet the specific requirements. Moreover, the porous structure can provide a good biological fixation to the surrounding tissue through inducing bone tissue growing into the porous cell [11]. Several methods [12–15] have been reported to fabricate porous metallic biomaterials, which typically include selfpropagating high-temperature synthesis (SHS), hot isostatic pressing (HIP), space-holder sintering process (SSP) and other conventional sintering (CS). Aguilar Maya et al. [16] fabricated a porous Ti–10Zr–10Nb alloy by using space-
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holder sintering to reach a porosity level of 70 % and a porous size of 260 lm. The material exhibits a Young’s modulus of 3.9 GPa and a compressive plateau stress of 67 MPa. Sirikul et al. [17] also synthesized a porous NiTi alloy by SHS technique, which have a porosity of 42–58 % and pore sizes between 340 and 500 lm, and the open porosity was controlled in the range of 87–98 %. However, the currently available methods are still difficult to construct the good porous alloys for tissue ingrowth through controlling the pore sizes, pore structure and mechanical properties. In the present work, we introduce a porous Ti– Nb alloy fabricated by a method of integrating of the sponge impregnation and sintering technique. The characteristics of the porous Ti-Nb alloy and the mechanical properties were evaluated and the biological behaviour of the porous Ti–Nb alloy was assessed.
2 Experimental 2.1 Preparation and characterization of porous Ti– Nb alloy scaffolds
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The apparent porosity and the open porosity of the Ti– 25 wt%Nb alloy were measured by the Archimedes method, which is described in detail elsewhere [18, 19]. Compression tests for mechanical property evaluation were performed by using an Instron3369 Universal Electromechanical Testing System equipped with Bluehill software with 250 KN capacity at a constant crosshead speed of 0.5 mm/min. Young’s modulus and compressive strength were determined from the stress–strain plots derived from load–displacement data recorded during compression testing. The reported data were obtained from averaging the results of five samples. All the tests were performed at ambient temperature (*25 °C). The phase composition and crystallinity of Ti– 25 wt%Nb alloy samples were characterized by X-ray diffraction (XRD) analysis at room temperature. A Jeol6360LV scanning electron microscopy (SEM), equipped with an EDAX-Phoenix energy dispersive spectroscopy X-ray (EDX) analysis system to analyse elemental distribution of the porous Ti–25 wt%Nb alloy, was utilized to analysis cross sectional microstructures. 2.2 Isolation and culture of MSCs
The starting elemental metal powders of TiH2 (with a purity of 99.9 % and an average diameter of 45 lm), Niobiumhydrides powder (with a purity of 99.5 % and an average diameter of 9.97 lm) were weighed to give a nominal composition of Ti–25 wt%Nb. These powders were blended together by ball milling for 2 h in planetary ball mill at the rate of 200 rpm. A commercial polyurethane sponge (Dongguan Zhanyu Industry & Trade Co., Ltd., P. R. China) employed in the experiment has an interconnected macroporosity, with an open porosity of 50–80 pores per inch (PPI), which corresponds to pore diameters 500–850 lm. And the density of the sponge is in the range of between 0.05 and 0.07 g/cm3. Before impregnating mixed powder slurry into the sponge, the sponge was cut in blocks of A10 9 25 mm3 and was repeatedly washed in 10 % NaOH solution at the temperature of 40–60 °C for 30 min, then swilled with deionized pure water for six times. In order to control the slurry viscosity and optimize the ability of blended powders to coat the sponge, 5 % (wt%) polyving akohol(PVA)aqueous solution was used as a binder. The slurries were kept under constant magnetic stirring during 1 h, and then the specimens were carefully impregnated in the slurries. Subsequently, the specimens were dried in vacuum at 100 °C for 24 h. Finally, the sponge scaffolds and the PVA were burned out from the green body at 300 °C for 3 h and the subsequent sintering process was carried out in a high vacuum furnace (below 9.0 9 10-4 Pa) at 1350 °C for 2 h with the heating/cooling rate of 10 °C/min.
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Rabbit bone marrow mesenchymal stem cells (MSCs) were derived from healthy 2.4 kg 8-week old male New Zealand white rabbit. Rabbit bone marrow-derived MSCs were flushed out via aspiration from the femoral bone marrow iliac crest by using a 16-gauge syringe needle, collecting 3 ml of marrow blood into 100 u/ml of heparin (2 ml). This cell suspension was placed over a Ficoll solution (1.073 g/ ml) slowly. The marrow blood was filtered through a cell strainer to exclude any fatty tissues and blood clots, and processed in a centrifuge at 2000rmp for 15 min. Subsequently, nucleated cells at the medium-Percoll interface were collected, then washed with 10 % PBS medium (including penicillin 100 l/ml and streptomycin 100 lg/ml) and plated in culture flasks at 1.0 9 106/cm2 with complete medium. Culture flasks were maintained in a humidified inculcator at 37 °C with 5 % CO2. Adhered cells were fed by complete replacement of the medium per 3 days. When the layer was 80 % confluent, the culture was trypsinized and cells were subcultured onto two or three culture flasks at a cell density of 1 9 104 cells/cm2. Cells underwent three passages in culture to ensure that all contaminating hematopoitic cells had been totally washed out. Detached cells were washed and re-suspended in PBS, and incubated with the following mono clonal antibodies directly conjugated with fluorescein isothiocyanate (FITC): CD29, CD34, and CD44. Cells were then washed and immunophenotypic analysis was then performed by flow cytometry.
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2.3 Seeding of MSCs on porous Ti-Nb alloy surface
2.5 Statistical analysis
The specimens were cut into U10 mm 9 2 mm with electrical discharge wire cutting and gently polished with 1000# sand paper. Then, they were sterilized in a highpressure autoclave at 120 °C for 30 min. Following this, MSCS of the 3rd passage were then seeded onto sample surfaces and placed in a 12-well plate. The initial cell density was 1 9 105 cell per wall. 2 milliliters of Dulbeccos modified Eagles medium (DMEM) enriched with 10 % fetal bovine serum was added to each well. MSCs were stained with acridine orange after 3, 24 and 72 h of incubation to assess the adhesion and proliferation by fluorescence microscopy. And the cell-material complex of 72 h of incubation was isolated, fixed with 3 % glutaraldehyde for 30 min, dehydrated with ethanol and dried in vacuum. Dried samples were then gold-coated and observed under SEM.
All the data were analysed using SPSS (version 15.0) software for windows student version. The differences among the groups were analysed through one-way analysis of variance (ANOVA). A p value of less than 0.05 was considered to be statistically significant..
2.4 MTT assay 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was utilized to determine the viable cell numbers based on the reductive cleavage of MTT (a yellow salt) to formazan (a dark blue compound) by mitochondrial dehydrogenase of living cells. Cells were seeded in 96-well plates with cell seeding density of 1 9 105 in a humidified incubator containing 5 % CO2 at 37 °C for 24 h. The wells were divided into three groups for the following tests: (1) the porous Ti–25 wt%Nb alloy group, the cells were cultured in the extract of the porous Ti–25 wt%Nb alloy (extracted 1 g material with 5 mL medium); (2) the bulk Ti–25 wt%Nb alloy group, the cells were cultured in the extract of the porous Ti–25 wt%Nb alloy (extracted 1 g material with 5 mL medium); (3) negative control group, the cells were cultured in fresh RPMI-1640 medium. Then, after being incubated for 1, 2, 3 and 7 days, cells were incubated for another 4 h with 10 lmol/L of MTT (Thiazolyl Blue Tetrazolium Bromide; Gibco,USA), respectively. Washing with PBS was followed by the addition of DMSO(dimethyl sulfoxide; Gibco,USA) (150 lL) and gently shaking for 10– 15 min to ensure the complete dissolution. The optical density (OD) of the solution in each well was measured after 60 min of slow shaking the absorbance at a wavelength of 492 nm using a plate spectrophotometer. The relative growth rate (RGR) was calculated according to the following equation: RGR ¼ ODsamples =ODnegative control 100%
ð1Þ
The criteria for cell toxicity classification were defined as: RGR C 100 %, Level 0; RGR 75–99 %, Level I; RGR 50–74 %, Level II; RGR 25–49 %, Level III; RGR 1–24 %, Level IV; RGR 0, Level V.
3 Results 3.1 Material characterization The microstructure of porous Ti–25 wt%Nb alloy is shown in Fig. 1. It can be seen from Fig. 1a that pores on the surface of Ti–25 wt%Nb alloy showed a good 3D interconnected network with the pore size of between 300 and 600 lm, which was smaller than the size of sponge scaffolds (500–850 lm). Obviously, this is because the formation and the growth of sintering neck between powder particles make the origin pores shrink. During the process of impregnation, the sponge scaffold was impregnated into the blended suspension liquid. Simultaneously, in the sponge scaffold the skin of pores delivered the suspension liquid to different inter-connected surface of pores due to the drive of capillary force. And, subsequently, in the following high vacuum dry process, the water in PVA was evaporated; the powder particles were bonded together and stuck on the skin of pores. In the final sintering process the pore sizes came to shrink through gradual elimination of the sponge scaffold and mechanical bonding of solid particles. Figure 1b presents the morphology of human cancellous bone. It can be found that the porous structure of prepared highly porous Ti–25 wt%Nb alloy was similar to human cancellous bone. Moreover, it should be noted from Fig. 3a that there were many second pores distributed on the wall of the pores and their sizes of the pores are in the range of 5–20 lm, which could be attributed to the shrinkage of sintered particles. It was determined by Archimedes method that the porous Ti–25 wt%Nb alloy possessed a porosity of 71 ± 1.5 %. The porosity was a slight lower than that of the sponge scaffold. Moreover it is worth being pointed that the open porosity could achieve as high as 94 ± 1.3 %. The good interconnected network of sponge scaffold and considerable large pores (as high as 500-850 lm) ensured the carriers with powders could go along with skin of sponge pores rather than block them. XRD spectrum (Fig. 2) of the Ti–25 wt%Nb alloy, as expected, revealed that a ? b alloy and no other obvious impurities peak occurred. This indicates that during the sintering process the high vacuum (as low as 10-4 Pa) would ensure that the scaffold and the binder could be
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(a)
(b)
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Fig. 1 a SEM micrograph showing the morphology of pores in the Ti–25 wt%Nb alloy; b SEM micrograph showing the cancellous bone of human bone
(a) (a) Intensity/ a.u
--- α --- β
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Fig. 2 XRD spectrum of the porous Ti–25 wt%Nb alloy
76.95
At% 13.38 86.62
Correction
ZAF
Wt% 23.05
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burned out or volatilized effectively. Furthermore, the composition was confirmed by EDS, as shown in Fig. 3b, that Nb concentration was 23.05 wt%, approaching to the designed concentration of 25 wt%.
1.3 0.6 0.0
3.2 Mechanical properties Metallic biomaterials currently in use for medical applications are suffering from their higher elastic modulus than human bone (0.1–30 GPa) and lacking of sufficient osseointegration for implant longevity [20]. If the elastic modulus of those biomaterials is much greater than that of human bone, the bone-producing cells called osteoblasts are stimulated into generating more bone when the bone is stressed [21]. This will thereby further result in an occurrence of bone resorption, increasing the healing time and even finally implant surgery failure [22]. Therefore, the elastic modulus and the compressive strength of these biomaterials should be controlled in the range of human bone from 0.5 to 30 GPa and around 100 MPa, respectively [19]. In present work, the compressive strength and Young’s modulus of the porous Ti–25 wt%Nb alloy,
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Nb La
Ti La
Ti La
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4.00
5.00
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Fig. 3 a SEM micrograph showing the detailed morphology of porous wall surface of the porous Ti–25 wt%Nb alloy and b the EDS diagram and the analytic composition of metallic skeleton (marked by red square on SEM micrograph) of the Ti-25 wt%Nb alloy (Color figure online)
measured from the compression mechanical test, achieved 98.4 ± 4.5 MPa and 2.23 ± 0.5 GPa, respectively. 3.3 Cell culture and proliferation Figure 4 shows immunophenotypic analysis by flow cytometry to MSCS of the 3rd passage. It can be seen that CD44 were positively expressed in 96 %, CD29 in 95 %, but only CD34 in 5 % MSCs, which indicates that the 3rd passage of MSCs maintained standard immunophenotypy of MSCs.
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Fig. 4 Immunophenotypic analysis by flow cytome try to MSCS of the 3rd passage
Qualitatively, florescent optical micrographs of the adhesion and proliferation of MSCs on the porous Ti– 25 wt%Nb alloy are presented in Fig. 5. For better comparison, the bulk Ti–25 wt%Nb alloy was selected to assess its biocompatibility together with the porous Ti– 25 wt%Nb alloy. As seen, with increasing inoculation time, the adhesion and proliferation of MSCs on both materials showed a significant increase. Moreover, compared with the bulk Ti–25 wt%Nb alloy, the porous Ti– 25 wt%Nb alloy showed evidently much more MSCs on its surface. Furthermore, quantitatively, the total numbers of MSCs of the adhesion and proliferation on the both materials counted by fluorescence microscope is showed in Fig. 6. As expected, the adhesion and proliferation of MSCs on the porous Ti–25 wt%Nb alloy showed much more rapidly than that of the bulk Ti–25 wt%Nb alloy with increasing period of inoculation time from 3 to 72 h (P \ 0.05). Typically, the SEM morphology of MSCs on the porous Ti–25 wt%Nb alloy surface after 72 h is shown in Fig. 7. It can be seen that MSCs showed flatter or irregular morphology with the size of 5–20 lm, which partially covered
on the surface of porous Ti–25 wt%Nb alloy scaffold or extended inward the pores. The cells clustered and were abounded with pseudopodium, showing better adhesion and activity.
3.4 Cytotoxicity results In present study, the MTT assay was utilized to quantitatively determine the proliferation of the viable MSCs on both of the porous Ti–25 wt%Nb alloy and the bulk Ti– 25 wt%Nb alloy. Figure 8 shows a comparison of viable cell densities for the porous Ti–25 wt%Nb alloy, the bulk Ti–25 wt%Nb alloy and negative group after different cultured time of 2, 4 and 7 days. As seen, the optical density (OD) of the porous Ti–25 wt%Nb alloy is significantly higher than that of the bulk Ti–25 wt%Nb alloy and is just slightly lower than that of the negative group. Furthermore, the results of toxicity of both materials by MTT assay indicated that the RGR values of cells were in the range of between 94 and 113 %, indicating that porous materials were insignificant cytotoxicity (Level I).
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Fig. 5 Florescent optical micrographs showing the morphology of MSCs on the surface of bulk (a–c) and porous (d–f)- Ti–25 wt%Nb alloy cultured at different times, a and d 3 h, d and e 24 h, c and f 72 h 300
Bulk Ti-25 wt.% Nb alloy
Cell Number
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Porous Ti-25 wt.% Nb alloy
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Fig. 6 Cell numbers of the MSCs adhering on the bulk and porous Ti–25 wt%Nb alloy at different times
20μm
Fig. 7 SEM micrograph showing the surface of the porous Ti– 25 wt%Nb alloy after culturing 72 h in ambient environment (25 °C)
4 Discussion 4.1 Biomechanical behaviour Just mentioned earlier, suitable mechanical properties, such as modulus and strength, are quite important. The modulus of the bulk bio-metallic materials is more than 40 GPa according to the recent reports [23–26]. The effective way to low down the modulus is applying porous design to the metal. But, on the other hand, the decreasing modulus always results in a rapid decrease of strength. Therefore, getting the balance of suitable modulus and compressive
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strength for the porous alloy is critical. Reviewing our previous results [18, 19], as presented in Fig. 9, it can be found that the present porous Ti–25 wt%Nb alloy have the optimum compressive strength, which is higher than that of the porous Ti-50Ni alloy [19] and the porous Ta [18]. And the compressive strength is in the range of between cancellous bone and corticall bone [19], and met the mechanical properties of requirement of trabecular bone well. On the other hand, the elastic modulus of the porous Ti–25 wt%Nb alloy, similar to the porous Ti–50Ni alloy and the porous Ta are falling into the range of between
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Optical density
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Bulk Ti-25 wt.% Nb alloy Porous T- 25 wt.% Nb alloy Negative group
0.9 0.6 0.3 0.0
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Fig. 8 MTT assay of the bulk and porous Ti–25 wt%Nb alloys cultured at different times
Compressive strength /MPa
200 4.4~28.8 GPa
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0.50 GPa 3.99
0.07 GPa 2.21
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Ti-25Nb
Ti-50Ni
Ta
Cancellous bone
Corticalbone
Fig. 9 The compressive strength of the different alloys made by the same process with the same volume fraction of pores at a level of 70 %
cancellous bone and corticall bone. This fact is very attractive for its potential to minimize the stress shielding effect and to optimize short term and long term fixation, indicating that the porous Ti–25 wt%Nb alloy have a good biomechanical compatibility. 4.2 Biocompatibility behaviour Structurally, for a successful biometal porous implant, many aspects take important roles. First of all, the pore parameters should have specific range for in-growth of human bone tissues. Many previous studies [22, 27] have pointed out that many parameters, such as porosity, porous size and morphology, have great effects on the ability of interaction with cells. Porous Ti alloys are expected to offer better surface interaction area with cells by offering mechanical anchoring sites, in turn, facilitating the growth of cells [23]. Furthermore, an increasing number of investigations identified that the pore size of 100–600 lm can more easily enhance the bone tissue ingrowth and eventually good implant fixation. Our previous results [18,
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19] also revealed that the size of 300–600 lm of Ta and TiNi alloy can evidently promote the cells to ingrowth. Another important pore parameter is open porosity. As we know, the connectivity of porous material is extremely helpful. Because there are much more connected channel for adhesion, proliferation and differentiation when cells are attached to the material surface, as well as offering more connected channels to allow nutrient delivery, and waste removal. In the present study, the prepared porous Ti–25 wt%Nb alloy showed good pore structure mimicking human bone with 3D interconnected porous network of microscale (Fig. 1b), which can meet the requires mentioned above. As well as our previous investigations, the second pores in the range of 5–20 lm can offer a considerably good fixation of earlier cell attachment due to bigger surface area and roughness of pore surfaces, which is in according to some other findings [7, 28, 29]. Compositionally, the chemical components of the implant alloy play a significant role in determining the interaction between implant materials and their surrounding tissues. For a long term, spontaneously formed oxides whose compositions are Ti2O and Nb2O5, will provide a bio-inert layer on the alloys surface in the complex human body fluids [4]. Thereby, the enrichment of TiO2 and Nb2O5 on the surface suppressed the dissolution of Ti and Nb as ions. So, the Ti–25 wt%Nb alloy show high cell viability during the MTT assays.
5 Conclusions A high porosity of 71 %, with a 3D interconnected open porosity of 94 %, Ti–25 wt%Nb alloy was successfully prepared in combination of the sponge impregnation technique and sintering technique. This material can be capable of having a pore size of 300–600 lm, a compressive strength of 98.4 ± 4.5 MPa and compressive Young’s modulus of 2.23 ± 0.5 GPa. The Ti–25 wt%Nb alloy processes a ? b alloy, whilst the elements of Ti and Nb distributed uniformly and no obvious occurs during the preparation. The in vitro results revealed the highly porous Ti– 25 wt%Nb alloy have good biocompatibility, which can evidently promote adhesion, proliferation of MSCs. It is indicated that this porous Ti–25 wt%Nb alloy can induce steoblasts to penetrate into through the pores and interconnected channels, offering good biological fixation. All these data provide proofs further that porous Ti–25 wt%Nb alloy could be used as a good biomaterial in tissue engineering. Acknowledgments This work was supported by National Natural Science Foundation of China (No. 51404302,51274247), National
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High Technology Research and Development Program of China (863 Program) (No. 2012BAE06B00). The author Hailin Yang also would like to acknowledge the financial support from the independent project of State Key Laboratory of Powder Metallurgy Research Institute.
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