Nanocrystalline Ti_49.2Ni_50.8 shape memory alloy as orthopaedic implant material with better performance

1. Introduction

Different from other biomedical metallic alloys, such as Ti and its alloys, Co-Cr alloys, stainless steels, Zr and its alloys, and Ta and Nb alloys [[1], [2], [3]], TiNi alloys, with their unique shape memory effects and superelastic properties, occupy an indispensable place in minimally invasive therapy. Till now, TiNi alloys have been widely employed for manufacturing various medical devices, including orthopaedic implants, dental materials, biliary stents and cardiovascular stents, septal occluders, guidewires, and so on. Yet the clinical practice still demands for further improvements in the strength, fatigue, corrosion resistance and biocompatibility of TiNi shape memory alloys [4]. Moreover, the TiNi alloys showed poor osteogenesis and osseointegration [5]. It has been reported that the grain size of the material normally plays the most significant and dominant role in determining the mechanical and muti-functional properties of bulk polycrystalline metallic materials [6,7]. Inspired by above facts, it is interesting to study if TiNi alloys with nano-sized grain can improve the abovementioned properties or not.

In the last decade, the development of ultra-fine grained and nano-grained (UFG/NG) materials has attracted particular attentions because of the improved mechanical and corrosion properties compared with the coarse grained (CG) materials at initial stage [8]. Severe plastic deformation (SPD) techniques, including equal channel angular pressing (ECAP), high pressure torsion (HPT) and accumulative roll bonding (ARB) are effective tools for fabricating UFG/NG metals and alloys [[9], [10], [11], [12]]. Besides, SPD approaches can render material with relatively higher strength and novel bio-functional properties. This particular combination cannot be provided by the traditional processing techniques [[13], [14], [15]]. Previous studies have mainly focused on the fabrication and characterization of the UFG/NG materials, including the investigation of their grain refinement, improved fatigue behavior, hardness, thermal stability, magnetic properties, etc [8,[16], [17], [18]]. Recently, the relationship of grain size and the biological properties has been studied. It has been reported that the UFG/NG material surfaces can enhance protein absorption, which is one of the major intermediaries for cell adhesion and functions and tissue growth. Increased surface area and reactivity are considered to be the main reason [19]. Besides, it has been shown that SPD methods can provide bulk/surface materials with superior bio-functions [20]. Furthermore, enhanced mechanical properties would guarantee the biosafety of the UFG/NG materials under physiological strains, and lower the possibility of failure due to low strength. The higher mechanical strength also provides the opportunity of downsizing the implants and medical devices, which is favorable for noninvasive surgery [19], and is exactly the goal that biomaterial researchers actively and continually pursue [21].

The aim of the present study is to develop a novel nanocrystalline Ti_49.2Ni_50.8 alloy via SPD process on microcrystalline Ti_49.2Ni_50.8 alloy and evaluate its cell and tissue responses as well as the bio-functionality by in vitro and in vivo studies.

2. Materials and methods

2.1. Materials preparation

The commercial binary Ti_49.2Ni_50.8 alloy shape memory alloy was purchased from the Trillion Metals Co., Ltd., China, and used as microcrystalline control group. Nanocrystalline T Ti_49.2Ni_50.8 alloy samples were prepared by ECAP for 8 passes, with regime Bc route at 450 °C.

Experimental samples (disc shape with the size of φ4 mm × 1 mm) for in vitro study were mechanically polished up to 2000 grit, ultrasonically cleaned in acetone, absolute ethanol and distilled water in sequence, and then dried in open air. For cell experiment, all experimental samples were sterilized in an autoclave. The implants with the size ofφ4 mm × 7 mm were chosen for animal study. Before implantation, samples were sterilized by Gamma radiation.

2.2. Materials characterization

Transmission electron microscopy (TEM, JEM 200CX, JEOL, Japan) was employed for the identification of the phase constitution and morphology in the nanocrystalline Ti_49.2Ni_50.8 alloy.

2.3. Haemocompatibility assessment

The haemocompatibility assessment was in accordance with our published protocols [22], which is detailed as follows: for the hemolysis tests, healthy human blood was diluted with saline, followed by dipping the samples in 10 mL saline. Then 0.2 mL of diluted blood was added to the samples and the mixtures were incubated for 1 h at 37 °C. After that, the samples were centrifuged and the supernatant was carefully removed and transferred to 96 well plates for further analysis at 545 nm using microplate reader (Bio-RAD680). For the platelet adhesion test, the platelet-rich plasma (PRP) was prepared and then moved to the sample surfaces and incubated at 37 °C for 1 h. The samples were rinsed with PBS to remove the non-adherent platelets. After that, the adhered platelets were fixed in 2.5% glutaraldehyde solutions for 1 h at room temperature followed by dehydration in a gradient ethanol/distilled water mixture (50%, 60%, 70%, 80%, 90%, 95% (two times), and 100%). The platelet was observed by environmental scanning electron microscopy (ESEM, AMRAY-1910FE). Six different areas were randomly counted and values were expressed as the average number of adhered platelets per mm² of surface.

2.4. Protein adsorption

Bovine serum albumin (BSA, Baosai Biotechnology Inc., Beijing) was chosen as the standardized protein. The protein solution (100 μl, 0.2 mg/ml) was pipetted onto the sample surfaces and after incubation for 2, 4 and 24 h at 37 ℃, the non-adherent proteins were removed and mixed with micro-bicinchoninic acid (BCA) at 37 °C for 30 min. Finally, the albumin (both the removed albumin and the total albumin) was quantified using a microplate reader (Bio-RAD 680) at 570 nm.

2.5. Cell culture

Murine fibroblast cell lines (L-929) and osteoblast cell lines (MG63) were adopted in the present study to evaluate the cytocompatibility of samples. All the cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM), mixed with 10% fetal bovine serum (FBS), 100 U mL^-1 penicillin and 100 μg mL^-1 streptomycin at 37 ℃ in a humidified atmosphere of 5% CO₂.

2.6. Cytocompatibility evaluation

The cells were co-cultured with the nanocrystalline Ti_49.2Ni_50.8 alloy and commercial microcrystalline Ti_49.2Ni_50.8 alloy extracts, respectively. The control groups involved the use of DMEM medium as negative control and DMSO as positive control. After incubating the cells in a humidified atmosphere with 5% CO₂ at 37 ℃ for 1, 2, and 4 days, 10 μL MTT was added to each well. The samples were incubated with MTT for 4 h at 37 ℃, then 100 μL formazan solubilization solution (10% SDS in 0.01 M HCl) was added in each well overnight in the incubator in a humidified atmosphere. The spectrophotometric absorbance of the samples was measured by microplate reader (Bio-RAD680) at 570 nm with a reference wavelength of 630 nm.

2.7. Cell differentiation and mineralization

The osteoblasts’ alkaline phosphatase (ALP) activity was examined after 7, 14, and 21 days’ cultivation. After seeding cells onto the sample substrate for 7, 14 and 21 days in 96 well plates, the 96 well plates were incubated with solution of 200 μL 0.2% Triton X-100 for cell lysis. After 30 min’ cell lysis, the ALP activity was evaluated as the amount of nitrophenol released through the enzymatic reaction. The absorbance was measured at 405 nm using an ELISA reader (Bio-RAD680).

The cell mineralization was measured by the Alizarin Red S-stain. The mineralization was identified as the absorbance of calcium-bound Alizarin Red at 570 nm via an ELISA reader (Bio-RAD680).

2.8. Animal testing

12 months Beagle dogs were chosen for the animal study, with the approval of Institutional Animal Care and Use Committee (IACUC) of Peking University, China (approval No. 621.2531.31-14-01). The dogs were well fed and carefully observed for a week in Animal Care Center before implantation. The Beagle dogs were divided into five groups randomly during the animal study.

The surgical procedure includes anesthetization with 1 mL/kg pentobarbital solution. After pre-operative skin preparation, the nanocrystalline Ti_49.2Ni_50.8 alloy samples were implanted into the right tibia of the Beagle dogs, while the commercial microcrystalline Ti_49.2Ni_50.8 alloy samples were implanted into the corresponding left tibia. The samples were harvested and fixed with 10% buffered formalin after 1, 2, 4, 8, and 12 weeks (n = 4) implantation. Specimens were dehydrated and then embedded in light-curing epoxy resin without decalcification. Histological sections were stained with toluidine blue, followed by observation on an optical microscope (Olympus IX71, Japan).

2.9. Micro-CT scanning and analysis

For the micro-CT scanning and analysis, a micro-CT imaging system (Skyscan 1072, Kontich, Belgium) with a high resolution of 18-μm-voxel was used. After scanning, the CTAn V1.7, NRecon V1.4, and CTVol V1.11 (SkyScan, Kontich, Belgium) analysis softwares were utilized for the final 3D-reconstruction images and bone tissue parameters.

2.10. Statistical analysis

Statistical analysis was performed with SPSS 18.0 software. Differences between groups were analyzed using one-way ANOVA, followed by post-hoc Tukey’s test. p value <0.05 was considered statistically significant.

The methods were carried out in accordance with the approved guidelines. All experimental protocols were approved by the Institutional Ethics Committee of Peking University. Written informed consent was obtained from all subjects.

3. Results and discussion

3.1. Microstructure

Fig. 1 shows the grain size observation of the nanocrystalline and commercial microcrystalline Ti_49.2Ni_50.8 alloy samples. From Fig. 1(a), it can be seen that the commercial microcrystalline Ti_49.2Ni_50.8 alloy has a coarse grain structure and the grain size ranges from 10 to 20 μm. Fig. 1(b) and (c) shows the TEM image and the grain size distribution of the nanocrystalline Ti49.2Ni50.8 biomedical alloy, respectively. It is quite obvious that the grain size of the nanocrystalline Ti_49.2Ni_50.8 alloy is in the range of 150-250 nm, which is much smaller than that of the commercial microcrystalline Ti_49.2Ni_50.8 alloy alloy. Previous studies have shown that the nano/ultrafine grained alloys can significantly enhance the mechanical properties [23], corrosion resistance [24] and bio-functionality [7].

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Fig. 1.   Microstructure observations ((a) optical microscopy and (b) TEM) of the grain size of microcrystalline Ti_49.2Ni_50.8 alloy group (a) and nanocrystalline Ti_49.2Ni_50.8 alloy group (b); (c) the quantification of the grain size distribution of nanocrystalline Ti49.2Ni50.8 alloy group.

3.2. Hemocompatibility

Both the hemolysis rates for the nanocrystalline and microcrystalline Ti_49.2Ni_50.8 alloy samples are quite low (about 0.1%), much lower than the safety threshold of 5%. The number of adhered platelets on the nanocrystalline Ti_49.2Ni_50.8 alloy is lower than that of the microcrystalline Ti_49.2Ni_50.8 alloy (Fig. 2). The low hemolysis rate and less adhered platelets indicate the better haemocompatibility of nanocrystalline Ti_49.2Ni_50.8 alloy.

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Fig. 2.   Platelet number on the nanocrystalline and microcrystalline Ti_49.2Ni_50.8 alloy groups.

3.3. Protein adsorption

A nonspecific protein named albumin was adopted to evaluate the general protein adsorption as bioaffinity. And the adhesion of albumin on top of experimental samples within the initial 24 h was shown in Fig. 3. It can be seen from Fig. 3 that within the first 2 h, the plot of BSA adsorption keeps ascending quickly with the passage of time, then gets temporarily saturated at 4 h and finally reaches the peak value till 24 h for the nanocrystalline Ti_49.2Ni_50.8 alloy group, the tissue culture plate and the microcrystallline Ti_49.2Ni_50.8 alloy group. The BSA adsorption onto the substrate of the nanocrystalline Ti_49.2Ni_50.8 alloy is slightly higher than that of the other two groups, although there is no statistical difference among the three groups. As the proteins are present in the extracellular matrix, cytoskeleton and membrane, they are generally involved in cell-substrate interactions and consequently influence cell adhesion, growth and differentiation [25].

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Fig. 3.   Albumin adsorption of nanocrystalline and microcrystalline Ti_49.2Ni_50.8 alloy groups.

3.4. Cell response

Fig. 4 shows the relative cell viability of both nanocrystalline and microcrystalline Ti_49.2Ni_50.8 alloy groups. The cell viability of the nanocrystalline Ti_49.2Ni_50.8 alloy group is higher than that of microcrystalline Ti_49.2Ni_50.8 alloy group, indicating the enhanced cell compatibility of Ti_49.2Ni_50.8 alloy by nanocrystallization.

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Fig. 4.   Cell viability of nanocrystalline and microcrystalline Ti_49.2Ni_50.8 groups, (a) MG63; (b) L929 (single asterisk ★ indicating p < 0.05 compared with negative cell control group; double asterisks ★ ★ indicating p < 0.05 compared with microcrystalline Ti_49.2Ni_50.8 group).

Fig. 5 demonstrates the ALP activity (a) and Alizarin Red staining (b) of both nanocrystalline and microcrystalline Ti_49.2Ni_50.8 alloy groups. The ALP activity for the nanocrystalline Ti_49.2Ni_50.8 alloy group demonstrates much promoted expression and developing activity over that of microcrystalline Ti_49.2Ni_50.8 alloy group (p < 0.05). Moreover, after 21 days’ cell culture, matrix mineralization on the nanocrystalline Ti_49.2Ni_50.8 alloy group shows a significantly higher alizarin red release than that of microcrystalline Ti_49.2Ni_50.8 alloy group (p < 0.05). The enhanced ALP activity and mineralization indicate the better osteogenesis functions of Ti_49.2Ni_50.8 alloy after nanocrystallization.

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Fig. 5.   (a) ALP activity and (b) Alizarin Red staining of nanocrystalline and microcrystalline Ti_49.2Ni_50.8 alloy groups (single asterisk ★ indicating p < 0.05 compared with negative cell control group; double asterisks ★ ★ indicating p < 0.05 compared with microcrystalline Ti_49.2Ni_50.8 alloy group).

3.5. In vivo biocompatibility

Fig. 6 demonstrates the micro-CT analyses of bone mineral density (BMD) (a), bone volume/tissue volume (BV/TV) (b), number of trabecular bone (Tb.N) (c), and the thickness of trabecular bone (Tb.Th) (d) around the implant from pre-operation up to 12 weeks post operation. The remarkably higher amount of BMD, BV/TV, Tb.N and Tb.Th around the nanocrystalline Ti_49.2Ni_50.8 alloy implant can be clearly revealed in comparison to that of the microcrystalline Ti_49.2Ni_50.8 alloy group during the whole 12 weeks operation period. This would further confirm the enhanced osteogenesis of Ti_49.2Ni_50.8 alloy after nanocrystallization, which is consistent with the abovementioned histological analyses.

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Fig. 6.   (a) Bone mineral density (BMD), (b) bone volume/tissue volume (BV/TV), (c) number of trabecular bone (Tb.N), and (d) thickness of trabecular bone (Tb.Th) around the implant till 12 weeks post operation (asterisk ★ indicating p < 0.05 compared with microcrystalline Ti_49.2Ni_50.8 alloy group).

Fig. 7 shows the histological images of bone tissues around the microcrystalline Ti_49.2Ni_50.8 alloy (a, c) and the nanocrystalline Ti_49.2Ni_50.8 alloy (b, d) implants at first 1 week (a, b) and 2 weeks (c, d). In the histological images, black area represents the implants; dark blue area represents the newly formed bone and normal blue stands for the original bone. As demonstrated in Fig. 7(a) and (b), at the first week, it can be observed that the osteocytes get together and the new bone starts to form (darker blue area) between the implant (dark area) and original bone (normal blue area). For the nanocrystallized TiNi alloy implant, the amount of newly formed bone is higher than that of microcrystalline Ti_49.2Ni_50.8 alloy group. Some of them even stretched out onto the gap or surface around the implant thread. In 2 weeks postoperatively (Fig. 7(d)), the nanocrystalline Ti_49.2Ni_50.8 alloy group’s higher ability of new bone formation than that of the microcrystalline Ti49.2Ni50.8 alloy group has been further proved with the evolutionary change of the shape and density of the freshly formed new bone. The new bone amount in the nanocrystalline Ti_49.2Ni_50.8 alloy group is profoundly increased than that at the first week. The new bone formation and integration of the nanocrystalline Ti_49.2Ni_50.8 alloy group is much better than that of the microcrystalline Ti_49.2Ni_50.8 alloy group.

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Fig. 7.   Histological images of bone tissues around the microcrystalline (a, c) and the nanocrystalline (b, d) Ti_49.2Ni_50.8 alloy implants at first 1 (a, b) and 2 (c, d) weeks.

Fig. 8 shows the histological images of bone tissues around the microcrystalline (a, c) and the nanocrystalline Ti_49.2Ni_50.8 alloy (b, d) implants at the follow-up 4 (a, b) and 8 (c, d) weeks. It can be seen that with the in vivo implantation time going on, the gaps between the implant and the original bone have been gradually filled up by the compact bone, especially for the nanocrystalline Ti_49.2Ni_50.8 alloy group (Fig. 8(b and d)).

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Fig. 8.   Histological images of bone tissues around the microcrystalline (a, c) and the nanocrystalline (b, d) Ti_49.2Ni_50.8 alloy implants at the follow-up 4 (a, b) and 8 (c, d) weeks.

Fig. 9 shows the histological images of bone tissues around the microcrystalline Ti_49.2Ni_50.8 alloy (a) and nanocrystalline Ti_49.2Ni_50.8 (b) alloy implants after 12 weeks’ implantation. As shown in Fig. 9, after 12 weeks’ implantation, newly formed bone has completely wrapped the implants. For the nanocrystallized Ti_49.2Ni_50.8 alloy group, symbols of osteogenesis and osteointegration have been verified by the direct bone-implant contact. However, on the other hand, fibrous tissue capsules (yellow arrow) have been observed for the microcrystalline Ti_49.2Ni_50.8 alloy group, which indicates its inferior osteogenesis and osteointegration compared with that of the nanocrystallized group.

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Fig. 9.   Histological images of bone tissues around the microcrystalline (a) and the nanocrystalline (b) Ti_49.2Ni_50.8 alloy implants after 12 weeks’ implantation.

It is well known that traditional metallic alloys usually have poor bone bonding and osteogenesis and this may lead to the implant loosening or even further premature failure after operation [26,27]. Previous studies have shown that the grain size of metallic biomaterials has significant effect on absorption of proteins [28], which would mediate cell adhesion and enhance cell functions and tissue growth. The TEM analysis demonstrates that the grain size of the nanocrystalline Ti_49.2Ni_50.8 alloy is in the range of 150-250 nm, which is way smaller than that of the commercial coarse grained Ti_49.2Ni_50.8 alloy(10-20 μm). The nano-scale grain size of the nanocrystalline Ti_49.2Ni_50.8 alloy provides the material with improved bio-functionality, including enhanced cell growth and proliferation, increased bone adaptation, osteogenesis and osteointegration. Relative increase in surface area and enhanced biochemical reactivity are the distinctive aspects of nanostructured metallic biomaterials, which guarantee them having great potential to further manipulate their interactions with cells and tissues [19,29].

4. Conclusions

In the present study, biomedical nanocrystalline Ti_49.2Ni_50.8 alloy has been developed via ECAP. The enhanced cell viability, adhesion, proliferation, ALP activity and mineralization indicate the better osteogenesis functions of Ti_49.2Ni_50.8 alloy after nanocrystallization. The periphery thickness of new bone in the nanocrystalline Ti_49.2Ni_50.8 alloy group was increased. The new bone formation and integration of the nanocrystalline Ti_49.2Ni_50.8 alloy group is better than that of the microcrystalline Ti_49.2Ni_50.8 alloy group, indicating the nanocrystalline Ti_49.2Ni_50.8 alloy is a promising candidate as potential orthopaedic biomaterials with better biocompatibility.

Acknowledgements

This work is jointly supported by the National Key R&D Program of China (No. 2018YFC1106600), National Natural Science Foundation of China (NSFC) and the Russian Foundation for Basic Research (RFBR) NSFC-RFBR Cooperative Project (No. 51611130054), the National Natural Science Foundation of China (Nos. 51431002 and 51871004), and the National Natural Science Foundation of China (NSFC) and the Research Grants Council (RGC) of Hong Kong NSFC-RGC Joint Research Scheme (Grant No. 5161101031). R. Z. Valiev gratefully acknowledges the financial support from Saint Petersburg State University in the framework of Call 3 project (id 26130576).