Fabrication of Bioactive Titanium with Controlled Porous Structure and Cell Culture in Vitro

Rare Metal Materials and Engineering Volume 39, Issue 10, October 2010 Online English edition of the Chinese language journal ARTICLE

Cite this article as: Rare Metal Materials and Engineering, 2010, 39(10): 1697−1701.

Fabrication of Bioactive Titanium with Controlled Porous Structure and Cell Culture in Vitro Li Xiang1, 1

Wang Chengtao1,

Wang Lin2,

Zhang Wenguang1,

Li Yuanchao1

2

Shanghai Jiaotong University, Shanghai 200240, China; Xijing Hospital, The Fourth Military Medical University, Xi’an 710023, China

Abstract: One of the direct metal forming techniques, electron beam melting (EBM) process, was used to fabricate Ti6Al4V implants with controllable porous structure. The micro-structural pore characterization, porosity and mechanical properties of the fabricated implants were investigated. Scanning electron microscope (SEM) observation shows that the porous structure of fabricated samples coincide with the designed architecture. It is demonstrated that EBM process can provide accurate control over the internal pore architectures of the implant. The compressive strength of the implant with porosity of 60.1% is 163 MPa. The Young's modulus is 14 GPa, which is similar to that of cortical bone. The surface modification by improved alkali-heat treatment induces apatite formation in simulated body fluid (SBF). In vitro cell culture experiment results reveal that osteoblasts will spread and proliferate on the surface of modified specimens over a culture time of 7 d. Key words: electron beam melting; Ti6Al4V; porous structure; bioactivity; cell culture

Titanium and its alloys are widely used in orthopedic and dental implants thanks to their excellent mechanical properties, biocompatibility and good corrosion resistance. However, a major problem concerning metallic implants in orthopaedic surgery is the mismatch of Young’s modulus between the natural bone (10-30 GPa) and bulk metallic biomaterials (110 GPa for Ti). Due to this mechanical mismatch, the bone is insufficiently loaded and subjected to the stress shielded, which can lead to bone resorption and eventual loosening of the implant. A suggestion to overcome this drawback could be the use of porous materials. Porous metallic materials are increasingly attracting the widespread interest of researchers as a method of reducing mechanical mismatches and achieving stable long-term fixation by means of full bone ingrowth[1-3]. A number of processes have been developed for producing porous titanium and its alloy implants, including powder sintering approach, combustion synthesis, plasma spraying and polymeric sponge replication. However, these conventional techniques have limitations in the control over the external shape as well as internal pore architecture of the implants. Rapid prototyping (RP), or solid freeform fabrication (SFF), is a common name for a group of techniques that can generate

a physical model directly from computer-aided design (CAD) data. It is an additive process in which each part is constructed in a layer-by-layer manner. Direct metal forming technology is a hot topic in the RP field[4-6]. In this process, metal powders are used as starting materials, and RP route is applied to produce metal components. The medical implants produced by direct metal RP technology can be tailored to have anatomical shapes and controlled pore architectures, including pore shape, size, distribution, orientation and interconnectivity. Xue and Krishna et al[7, 8] reported that porous titanium implants were fabricated using laser engineering net shaping (LENSTM), which showed tailored porous structure, good mechanical properties, and biocompatibility. Direct laser forming (DLF) was utilized to produce individually structured Ti6Al4V by Hollander et al[9]. The pore sizes were reduced by the DLF processing by approximate 300 µm. The osteoblastic cells spread and proliferated on DLF-Ti6Al4V over a culture time of 14 d. Due to the bioinert nature, titanium and its alloys are generally encapsulated by fibrous tissue after implantation into the living body. One of the effective methods is to modify the titanium implant surfaces to be bioactive. The aim of this study

Received date：October 10, 2009 Corresponding author: Li Xiang, Ph. D., School of Mechanical Engineering, Shanghai Jiaotong University, Shanghai 200240, P. R. China, Tel: 0086-21-34206815, E-mail: [email protected] Copyright © 2010, Northwest Institute for Nonferrous Metal Research. Published by Elsevier BV. All rights reserved.

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is to fabricate Ti6Al4V implants with controlled porous structure using electron beam melting (EBM) process and to modify the implant surfaces using alkali-heat treatment. The porous structures and mechanical properties of the prepared implants were characterized. The surface bioactivity was tested by immersion in simulated body fluid (SBF). The biological property was evaluated by in vitro cell culture.

1 Experimental Ti6Al4V powder with an average particle size of 50 μm (Arcam AB, Sweden) was used in this study. The chemical composition of the powder is shown in Table 1. The shape of Ti6Al4V powder was analyzed by scanning electron microscope (shown in Fig. 1). The EBM machine is EMB-S12 (Arcam AB, Sweden). Scanning electron microscopy (SEM, JSM-6460, JEOL, Japan) was used to examine the surface morphology of porous Ti6Al4V implants. The porosity of porous implants was evaluated from the mass and the apparent volume of the samples (n=5). The following calculation was made: 1−ratio of the mass of the porous implant to the mass of a dense implant with the same volume. Compression tests were conducted with a MTS 810 material testing system using a 10-kN load-cell. Values of peak stress, 0.2%-offset yield stress and Young’s modulus were calculated for each sample. The crosshead speed was set at 0.5 mm/min. Chemical reagents used in this experiment were analytically pure grade, as follows: NaOH, HCl(36.5%), NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, CaCl2, Na2SO4 and tris-hydroxymethyl aminomethane. Simulated body fluid (SBF) was prepared according to Kokubo’s method[10]. The porous structure was designed with a commercial CAD software (Unigraphics NX, EDS). The design was a cylinder (diameter 11.6 mm, height 16.8 mm) shown in Fig. 2a. The pore size was about 800 μm. The distance between layers was 1.2 mm. After the CAD data of the structures were converted Table 1

Composition of Ti6Al4V powder (mass fraction, %)

Element

Al

V

Fe

Content

6.0

4.0

0.1

O

N

C

H

Ti

0.15 0.01 0.03 0.01 Balance

50 µm Fig.1 Morphology of Ti6Al4V powder

into STL data, which were imported into Materialise's Magics software, and converted into input file for EBM, the samples were produced on an Arcam’s EBM machine (EBM S12, Arcam AB, Sweden). Arcam’s EBM process is a direct metal layered fabrication technique, similar to rapid prototyping technologies such as stereolithography. EBM process begins with a CAD design file of a three-dimensional component, which is saved as STL data. The STL data is imported by the software that controls the EBM machine and converted into 2D sliced data for EBM process. The Ti6Al4V plate was heated with the electron beam with a maximum power of 4 kW in a bed of Ti6Al4V powder in a vacuum chamber. A layer of powder with 0.05 to 0.20 mm thick ness was then added onto the plate and a computer-controlled electron beam scanned the surface, selectively melting the powder. A new layer of powder was applied over this layer. Each layer was first preheated by scanning the beam at low power and then sintered at high velocity. The electron beam was scanned over the surface in two fixed scanning directions, perpendicular to each other, and in a predetermined pattern in order to distribute the heat as evenly as possible. The process was repeated to form eventually a part. After EBM process, the samples were left in the insulating powder bed in the vacuum chamber to achieve a slow cooling rate. The samples were cooled down to 150 oC in 11 h, then taken out of the vacuum chamber, and cooled down to room temperature in air. No subsequent heat treatment was applied. The modification method was according to Ref[11]. All samples were washed with pure acetone and ultra-pure water in an ultrasonic cleaner, and then immersed in 30 mL of 10 mol/L NaOH aqueous solution at 60 °C for 24 h. After the treatment, the specimens were gently washed with ultra-pure water, and then immersed in 30 mL of ultra-pure water at 40 °C for 48 h. Subsequently, samples were soaked in 0.5 mol/L HCl for 24 h. After washed with ultra-pure water, these samples were heated up to 600 °C at a rate of 5 °C/min in an electric furnace, kept for 1 h, and then allowed to cool in the furnace. The bioactivity of chemically pretreated samples was evaluated in terms of apatite-forming ability by soaking the samples in SBF solution under static conditions at 37 °C for 3, 7, and 14 d. The solution was changed every 2 d to maintain the concentrations of the solution. The modified surfaces were examined by scanning electron microscopy equipped with EDX analysis and X-ray diffractometry (XRD). All samples were sterilized in ethylene oxide gas. Osteoblastic cells were isolated from new born New Zealand rabbit’s calvaria. The third passage cells were used in the present study. 2×106 cells were seeded onto each specimen in a drop-wise manner. After covered with medium, the cultures were incubated at 37 °C in a 5% CO2, 100% relative humidity incubator. The medium was changed every 2 d. The samples were removed from culture and washed with PBS after 3 and 7 d incubation. Fixation was carried out for overnight at 4 °C

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in 2% v/v glutaraldehyde in 0.1 mol/L sodium cacodylate buffer. The samples were washed in this buffer, dehydrated through an ethanol series, critical-point dried, sputtered with gold, and examined by SEM for cell morphologies. The samples for histological study were fixed in 10% formalin for 7 d, dehydrated through an ethanol series, cleared with toluene, and embedded in methylmethacrylate. After polymerization, thin sections were prepared with a modified sawing microtome technique. The sections were stained with Haematoxylin and Eosin (H&E) and examined with a standard microscope (Leica).

2

Results and Discussion

2.1 Characterization Fig. 2 shows the porous Ti6Al4V implant with orthogonal structure. It can be seen that the produced samples have the same structure with the design. It is indicated that EBM process has the capability to control the external shape and internal pore architectures of Ti6Al4V implants. Fig. 3 shows the SEM image of the porous samples. The surfaces of the inner pore walls are rough. The rough surface would be beneficial to cell attachment. The average pore size is (700 ± 80) μm measured from SEM images, which is close to the designed size. The porous structure with fully interconnected network would allow the ingrowth and revascularization of the tissue. Five samples were used for their mass and volume measurement. The result of volume/mass analysis reveals that the part porosity is (60.1 ± 2.4)%. 2.2 Mechanical properties b

a

Three samples were used for the compression test. Fig. 4 shows the stress-strain curve of the porous implants. The porous Ti6Al4V samples possesses compressive yield strength of (138 ± 8) MPa. The ultimate strength of porous Ti6Al4V implants is (163± 11) MPa. Young’s modulus of porous Ti6Al4V parts is (14 ± 3.1) GPa. The compressive strength and Young’s modulus of human cortical bone are about 190 MPa and 10-30 GPa, respectively. It is revealed that the mechanical properties of the EBM produced Ti6Al4V with orthogonal structure are similar to those of cortical bone, which can reduce the mechanical mismatch between the implant and the bone tissue. 2.3 Bioactivity test Fig.5 shows the precipitate on chemically treated samples with subsequent soaking in SBF for 3, 7, 14, and 21 d. Few spherical precipitate is deposited on sample surfaces after immersion in SBF for 3 d (shown in Fig. 5a). The precipitate increases and evenly distributes on sample surfaces after immersion in SBF for 7 d (shown in Fig. 5b). After 14 and 21 d, the precipitate covers the entire surfaces (shown in Fig. 5c and Fig. 5d). EDX analysis reveals that calcium, phosphorus and oxygen appear on the surface. It can be concluded that Ca2+ and PO43ions are deposited on sample surfaces. Fig.6 shows the XRD patterns of the samples after chemical treatment and immersion in SBF for 14 d. After chemical treatment, rutile and anatase appear on the surfaces, as shown in Fig. 6a. As shown in Fig. 6b, the positions of these diffraction peaks indicate that the deposits are constituted of poor crystalline hydroxyapatite. The mechanism of apatite formation on sample surfaces is as following[10]: when samples are soaked in alkaline solution, a hydrated titanium oxide gel layer is formed on their surfaces. This gel layer contains a considerable amount of water or hydrated ions and hence is mechanically unstable. It is dehydrated and densified to form an amorphous alkali titanate layer and tightly bonded to the sample surfaces by heat treatment at 600 °C. When these alkali-heat treated samples are exposed to SBF, the alkali ions are released from the amorphous alkali titanate layer and hydronium ions enter into the surface layer,

Fig.2 CAD model of porous structure (a) and the fabricated sample (b)

200

Stress/MPa

150 100 50

500 µm

0

0

5

10

15

20

Strain/% Fig.3 SEM image of porous implant

Fig.4 Stress-strain curve

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b

a

d

c

2 µm

Fig.5 SEM images of the bioactively treated samples soaked for different time in SBF: (a) 3 d, (b) 7 d, (c) 14 d, and (d) 21 d

Intensity/a.u.

: Titanium : Apatite : Anatase : Rutile

b 100 µm

a 20

30

40

50

60 Fig.8 Histological section of porous bioactive titanium/cells con-

2θ/(º)

structs cultured for 7 d

Fig.6 XRD patterns of the samples after chemically treating (a) and subsequent immersing in SBF for 14 d (b)

resulting in the formation of a titanium oxide hydrogel layer. The released alkali ions increase the degree of supersaturation by increasing pH, and the titanium oxide hydrogel will induce apatite nucleation on its surface. Consequently, a large number of apatite nuclei are formed on the samples. The bioactive surface can improve the osteoconduction and osseointegration of the implants. 2.4 Cell culture Fig. 7 shows the SEM image of cells culture on samples for 3 d. Polygonal and spindle shaped cells attached and spread on porous surfaces. The cell sizes are about 10-30 μm. Extracellular matrix is formed on sample surfaces. Fig. 8 shows the H&E stained sections of the sample after 7 in vitro culture. The samples cultured in vitro demonstrate the layers of cells and extracellular matrix on the surface of the sample. Some cells were found growing into pores.

3 Conclusion 1) One of direct metal rapid prototyping techniques, EBM process, can be used to fabricate porous Ti6Al4V implants. The external shape and internal pore architectures are well controlled. The mechanical properties of the produced samples are similar to those of human cortical bone. 2) Chemical treatment can be used to modify porous implant surfaces. After immersion in SBF, the apatite is formed on sample surfaces. The modified samples have the good bioactivity. 3) Osteoblastic cells can be well attached, spread and proliferated on bioactive porous Ti6Al4V samples, which could be as a candidate of bone substitute.

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Fig.7 SEM image of cell morphology cultured for 3 d

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