In situ formation of bioactive calcium titanate coatings on titanium screws for medical implants

Abstract

The objective of this study was to improve the biocompatibility of titanium screws using a hydrothermal technique combined with chemical oxidation treatment. Bioactive calcium titanate (CaTiO₃) coatings were synthesized on titanium screws by alkali heat treatment without sintering. A cell-based experiment was conducted to evaluate cell attachment and proliferation on the surface of modified titanium screws. Cells grown on the CaTiO₃-coated surfaces (denoted CT-TI) had a higher proliferation rate than their counterparts grown on titanium screws without any surface modification (denoted TI-TI). The results indicated that the CaTiO₃ coatings improved the surface bioadhesion characteristics of titanium screws. The biocompatibility of CaTiO₃ coatings on titanium was also investigated by implanting titanium screws covered with CaTiO₃ coatings into living bone. The interface between the bone tissue and titanium screws was observed by environmental scanning electron microscopy 2, 4, 8 and 12 weeks after implantation. The boundaries between the bone tissue and the titanium screws coated with CaTiO₃ became indistinguishable more quickly than that between bone tissue and titanium screws without any surface modification. Thus, the biocompatibility of titanium screws coated with CaTiO₃ was higher than that of titanium screws without any surface modification. Therefore, the hydrothermal technique is an excellent surface-modification method to improve the biocompatibility of titanium screws. We have great confidence that these surface-modified titanium screws will be useful in in vivo bone.

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1. Introduction

Titanium and its alloys have been widely used as biomaterials because of their excellent mechanical properties and biocompatibility.^1,2 However, the biocompatibility of titanium and its alloys is lower than that of bioactive ceramics such as Bioglass,³ AW-glass ceramic,⁴ hydroxyapatite,⁵ β-tricalcium phosphate,⁶ titanium dioxide,⁷ and calcium titanate (CaTiO₃).⁸ Numerous surface modification techniques have been proposed to improve the biocompatibility of titanium. Methods that cover the titanium or its alloys with a bioactive ceramic coating have been widely recognized. Bioactive coatings have been formed on substrates by methods such as plasma spraying,^9,10 sputtering,¹¹ sol–gel^12,13 and hydrothermal techniques.^14–16 However, plasma spraying and sputtering generally use expensive equipment, and the sol–gel method requires strict control of the hydrolysis of its precursors. Compared with these methods, the hydrothermal technique is quite simple and attractive, involving a wet chemical process carried out at a relatively low temperature. Materials with various morphologies and crystallinities can be fabricated by the hydrothermal technique by controlling synthesis conditions such as temperature, treatment time and solution composition. Accordingly, the hydrothermal method may be a useful surface modification technique to improve the biocompatibility of titanium and its alloys.

Recently, the hydrothermal technique has been used for surface modification to design biomaterials. For example, Hamada et al.¹⁷ synthesized CaTiO₃ coatings on pure titanium substrates by hydrothermal treatment using an aqueous solution of calcium chloride or calcium oxide. Xin et al.¹⁸ obtained strontium titanate nanotube arrays on titanium by hydrothermal treatment of anodized titania nanotube arrays. Calcium ions in a titanium oxide layer can improve the bone integration of titanium implants.¹⁹ Simulation experiments of in vitro biological activity have indicated that covering titanium and its alloys with CaTiO₃ coatings is an effective method to enhance the biocompatibility of titanium alloy.^20,21 Moreover, Ergun et al.²² found that CaTiO₃ can improve the adhesion of osteoblasts. Thus, CaTiO₃ is considered a potential bioactive material and has been studied by many researchers.

Researchers have attempted to synthesize CaTiO₃ coatings on titanium substrates through hydrothermal techniques.²³ Synthesis of CaTiO₃ coatings typically requires high concentration of sodium hydroxide and subsequent calcination treatment. However, in our work, CaTiO₃ coatings were easily formed in a solution with relatively low concentration of sodium hydroxide and without subsequent calcination treatment. More gentle calcium acetate was employed to synthesize CaTiO₃ coatings. Also the in vitro and in vivo biological properties of the modified screws were systematically investigated. The results indicate that this method is projected to have immense potential in clinical medicine of bone implants.

2. Experimental

The study was approved by the ethics committee of the Hunan University and the third Xiangya Hospital of Central South University (approvl no. LLSC (LA) 2014-020) for biologic sample collection and animal procedures followed with NIH Guide for Care and Use of Laboratory Animals.

2.1 Materials and instrumentations

Titanium screws were purchased from Xinrong Best Medical Company, Suzhou, China. Acetone (≥99.5%), and sodium hydroxide (NaOH, ≥96.0%) were purchased from Guangdong Chemical Reagent Engineering Technological Research and Development Center, Shantou, China. Hydrogen peroxide (H₂O₂, ≥30%), calcium acetate (Ca(CH₃COO)₂H₂O, ≥96.0%) and nitric acid (HNO₃, ≥65%) were purchased from Hunan Jingke Technological Company, Changsha, China. All chemicals were used without further purification.

The surface morphology of specimens was observed by scanning electron microscopy (SEM, S-4800). The crystallinity of the CaTiO₃ coatings was characterized by XRD using Cu Kα radiation (λ = 1.54056 Å) in step-scan mode at a rate of 8° min⁻¹ and an X-ray incident angle α = 5.0° against the specimen surface (2500; Rigaku, Japan). The prepared bone specimens were observed by environmental SEM (FEI Quanta-200).

2.2 Preparation of CaTiO₃ coatings

Titanium screws (Ti–6Al–4V, φ 2.0 mm, 10 mm long) were ultrasonically washed with acetone and distilled water for 30 min, then dried at 353 K for 60 min. The cleaned screws were treated in 2 M H₂O₂/0.1 M HNO₃ aqueous solution at 353 K for 30 min. The screws were placed in a Teflon-lined autoclave (Keli, Yantai, China) filled with an aqueous solution of 1 M NaOH and 0.04 M Ca(CH₃COO)₂H₂O to 50 mL. The reactor was maintained at 383 K for 4 h and then air-cooled to room temperature. The titanium screws were successively washed with dilute HCl aqueous solution, and distilled water to remove any possible remaining ions until the solution pH was neutral. Finally, the titanium screws were dried in an oven (Yiheng, Shanghai, China) at 353 K.

2.3 In vitro biocompatibility properties

Studies have demonstrated that proliferation and differentiation of osteoblast cells are the essential steps that occur before bone mineralization.²⁴ Therefore, osteoblast mineralization, the proliferation ability and ALP activity of MC3T3-E1 cells cultured on titanium screws were investigated in this study. Before seeding the cells on biomaterials surfaces, the cells were revived.

2.3.1 Proliferation ability of MC3T3-E1 cells on modified titanium screws. To assess cell proliferation on the surface of the modified titanium screws, 1 × 10⁴ MC3T3-E1 cells were seeded on TI-TI and CT-TI titanium screws in a 24-well plate and allowed to adhere for 3 h. The cells were then cultured in a-minimum essential medium (a-MEM; Gibco, Grand Island, NY, USA and Hyclone, Logan, UT, USA). The nutrient solution was refreshed every 3 days. A 10 μL aliquot of 3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-di-phenytetrazoliumromide (MTT) (Amresco, Solon, OH, USA) solution was added to each well at the designated time points (1, 3 and 7 days). The cells were then incubated at 310 K for another 4 h. The samples were rinsed three times with phosphate-buffered saline (Dingguo Biotechnology, Beijing, China). To dissolve formazan crystals, 250 μL of dimethyl sulfoxide (Dingguo Biotechnology) was added to each well. The samples were then transferred to a fresh 96-well plate. The absorbance of these samples at 490 nm was measured with a spectrometer (DG3022A, Huadong Electronics, Nanjing, China).²⁵

2.3.2 ALP activity of MC3T3-E1 cells on the surface of modified titanium screws. To assess the ALP activity of the MC3T3-E1 cells grown on the surface of modified titanium screws, 1 × 10⁴ cells were seeded on each screw and cultured in a 24-well plate for 1, 3 and 7 days. At the predetermined time, the culture medium was poured out and the cells were washed three times with phosphate-buffered saline, followed by washing once in 50 mM cold tris buffer (Sinopharm Chemical Reagent Co., Shanghai, China). The cells were lysed in 200 μL of 0.1% Triton X-100 (Sinopharm Chemical Reagent Co.) for 12 h at 277 K. The ALP activity of samples was calculated using an automatic biochemistry analyzer (GF-2245; Jingke, Shanghai, China). Cell proliferation rate and ALP activity data were collected from three separate experiments and expressed as mean ± standard deviation. One-way ANOVA and Student–Newman–Keuls post-hoc tests were used to determine the level of statistical significance, and P < 0.05 was considered statistically significant.

2.3.3 Calcium nodule evaluation. All samples (CT-TI and TI-TI screws) were sterilized in steam autoclave and adjust the density of the cells to approximately 10⁴ mL⁻¹. Then, set the samples in a 24-well plate (one sample in each well) and add 1 mL cell suspensions into each well. The medium was refreshed every 3 days. After the stipulated time period (21 days), the samples were washed 3 times with phosphate-buffered saline (PBS), and then, cells were fixed for 4 h with 2.5% glutaraldehyde diluted in PBS. The cells adhered on the material surfaces were dehydrated using a series of ethanol solution (50, 70, 90, and 100%) for 10 min and then further dried at room temperature. The dried samples were sputter-coated with gold and examined by scanning electron microscope (SEM).

2.4 In vivo biocompatibility properties

2.4.1 Surgical operation. Forty-eight rabbits were anesthetized with 25% ethyl carbamate (Sinopharm Chemical Reagent Co.) (2 mL kg⁻¹). After general anesthesia, the rabbits were fixed on an operating table, epilated and operated upon under sterile conditions. Two holes were drilled in the left and right femoral condyles of each rabbit. Subsequently, titanium screws coated with CaTiO₃ and titanium screws without coatings were implanted into the holes randomly. After the wounds were washed, the ends of the nails were covered with soft tissue. After the operation, rabbits were free weighed, placed back in cages and given an intramuscular injection of 80 000 units gentamicin sulfate (Henan Topfond Pharmaceutical Co., Zhumadian, China) once each day for 3 days.

2.4.2 Preparation of bone specimens. The forty-eight rabbits were killed after being reared in cages for 2, 4, 8 and 12 weeks. Femoral condyles with titanium screw implants were taken out and fixed with 10 vol% formalin. The femoral condyles without decalcification were sliced into bone specimens with a hard tissue slicer (Leitz-1600; Leitz, Benzheim, Germany) as follows. First, the bone tissues were dehydrated three times with new acetone each time for 12 h. Then, the bone tissues were embedded in resin and cut into 30 μm-thick slices.

3. Results and discussion

3.1 Surface morphology of titanium oxide coatings on titanium screws

The surface morphologies of the titanium screws after chemical treatment with H₂O₂/HNO₃ aqueous solution are depicted in Fig. 1. The surfaces of the titanium screws were coated with titanium oxide after chemical treatment for 30 min, although many discontinuous microcracks were observed, as shown in Fig. 1(b). No exfoliation of the coatings was observed during treatment.

Fig. 1 SEM images of titanium oxide coatings on titanium screw substrates after chemical oxidation with H₂O₂/HNO₃ aqueous solution for 30 min. Magnification: ×3000 for (a); ×10 000 for (b).

3.2 Structure of CaTiO₃ coatings on the surface of titanium screws

3.2.1 Surface morphology. The surface morphologies of CaTiO₃ coatings fabricated on titanium screws are presented in Fig. 2. As illustrated in Fig. 2(a), the sample surface was coated with a dense layer of CaTiO₃ that contained some cracks. A highly magnified image (Fig. 2(b)) showed that the CaTiO₃ coating was composed of uniform cubic particles. The surface layer after immersion in the Ca-rich solution consisted of a porous hydrogel network structure containing many of small, spongy islands.²⁶ The result in the present work is quite different from that of previous work.

Fig. 2 SEM images of CaTiO₃ coatings on titanium screw substrates after hydrothermal treatment. Magnification: ×5000 for (a); ×20 000 for (b).

3.2.2 XRD analysis. X-ray diffraction (XRD) patterns of titanium screws before and after hydrothermal treatment are displayed in Fig. 3. As well as the peaks of the titanium substrate and titanium oxide, those consistent with CaTiO₃ can also be identified on the treated sample surfaces. Thus, a coating composed mostly of CaTiO₃ formed on the titanium screws after hydrothermal treatment. Synthesis of CaTiO₃ coatings typically requires high-temperature heat treatment. However, in the present work, CaTiO₃ coatings were easily obtained without calcination treatment.

Fig. 3 XRD patterns of (a) titanium substrate without any surface modification and (b) CaTiO₃ coatings after hydrothermal treatment.

3.3 Cell response to titanium screws

The proliferation of MC3T3-E1 cells cultured on the screws with and without CaTiO₃ at each designed time point is shown in Fig. 4. There was no significant difference between the two kinds of titanium screws on the first day (P > 0.05). However, the proliferation rate on the CT-TI screws was significantly higher than that on the TI-TI screws after 3 days (P < 0.05). The difference in the proliferation rates of the two kinds of titanium screws was even more obvious when the cells were cultured for 7 days. The alkaline phosphatase (ALP) activity of MC3T3-E1 cells cultured on CT-TI and TI-TI screws for 1, 3 and 7 days is presented in Fig. 5. Similar to the results obtained regarding cell proliferation rate, the CT-TI screws exhibited significantly enhanced ALP activity compared with that of the TI-TI screws (P < 0.05). These results indicate that the CT-TI screws are more conducive to cell attachment and proliferation than the TI-TI ones.

Fig. 4 The proliferation of MC3T3-E1 cells cultured on the TI-TI and CT-TI screws for 1, 3 and 7 d (d: days).

Fig. 5 The ALP activity of MC3T3-E1 cells cultured on the TI-TI and CT-TI screws for 1, 3 and 7 d (d: days).

In vitro osteoblasts culturing with the implants is one of the most widely used methods to evaluate biocompatibility. Calcium nodules are key indicators for osteoblast differentiation and osteoblast regeneration ability on surface of biomaterial.²⁷ As shown in Fig. 6, cell adhesion volume on CT-TI material surface increased significantly, and cells connected more closely than that on TI-TI material. Also it was observed that large amounts of calcium salts formed between cells on CT-TI material. However, there were only small amounts of calcium salts observed on TI-TI surface. Therefore, the result indicates that CT-TI screw is more conducive to osteoblasts osteogenesis and relevant osteogenesis mineralization than TI-TI screw.

Fig. 6 SEM images of cells on the surface of (a) TI-TI screw and (b) CT-TI screw.

3.4 The in vivo biocompatibility of titanium screws coated with CaTiO₃

The effect of CaTiO₃ coatings on the in vivo biocompatibility of titanium screws was observed using an environmental scanning electron microscope. As shown in Fig. 7, the interface between the host bone and CT-TI screws became less defined as the implanted time lengthened. In contrast, there were no distinct differences observed at the interface between the host bone and TI-TI screws implanted into rabbit femoral condyles after implantation for 2, 4, 8 and 12 weeks (Fig. 7(a), (c), (e) and (g), respectively). The interface between bone tissue and TI-TI screws had not obviously improved even after 12 weeks of implantation. Conversely, the interface between bone tissues and CT-TI screws improved after only 2 weeks of implantation (Fig. 7(b)). The boundary between the bone tissue and CT-TI screws became quite indistinct after 12 weeks of implantation (Fig. 7(h)). The CT-TI screws seemed to readily combine with the bone tissue, while the TI-TI screws did not. There are three possible reasons for this result. First, the CaTiO₃ coatings could promote bone cell adhesion and proliferation as showed by cell experiment in this work, and this is an essential step that occurs before bone mineralization. Second, the Ca-rich coating may combine with bone tissue by chemical bonding which is commonly stronger than physical bonding. Third, as shown in Fig. 2(a), the dense and rugged coating may increase the contact area with bone tissue substantially. Overall, Fig. 7 reveals that the CT-TI screws possessed better biocompatibility than the TI-TI ones.

Fig. 7 ESEM images of the bone specimens after the (a, c, e, g) TI-TI and (b, d, f, h) CT-TI screws were implanted into rabbits' femoral condyles for (a, b) 2 Ws, (c, d) 4 Ws, (e, f) 8 Ws and (g, h) 12 Ws (Ws: weeks).

4. Conclusions

To improve the biocompatibility of titanium, a facile hydrothermal technique combined with chemical oxidation was used to modify the surface of medical titanium. The results showed that crystalline CaTiO₃ coatings composed of uniform cubic particles were directly synthesized on titanium substrates without calcination. In vitro biocompatibility experiments showed that the CT-TI screws increased the proliferation and ALP activity of MC3T3-E1 cells more than TI-TI screws. In vivo experiments revealed that CT-TI screws could combine with bone tissue more effectively than the TI-TI screws. These findings indicate that the biocompatibility of titanium screws is distinctly improved by in situ coating with CaTiO₃. This simple and facile method may be a promising new technique to improve the biocompatibility of medical implants.

Acknowledgements

We gratefully thank the Natural Science Foundation of China (No. 81171461, No. 51402100), the Youth 1000 Talent Program of China, the Natural Science Foundation of Hunan Province (No. 11JJ4013, No. 13JJ2013), the Fundamental Research Funds for the Central Universities, and Fundamental Research Funds by the Central South University for financial support.

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Footnote

† Authors contributed equally to this work.

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