Enhanced in vitro bioactivity and osteoblast response of nanocrystalline titanium processed through ball milling.
The mechanical properties of titanium and its alloys are good enough for load bearing implants in medical applications besides being low density compared to other metallic biomaterials. Commercial pure titanium (cpTi) is a widely used material of choice for medical implants as they are chemically inert, biocompatible with human tissue, and resistant to corrosion by human body fluids . There is great interest in the formation of nanosize/ submicron particles due to their unusual properties as the morphology of the biomaterial is critical to its success as an implant material i.e. cells live in a nano-featured environment of a complex mixture of pores, ridges, and fibers of extracellular matrix (ECM). This information eventually leads to the concept of nano biomaterials with advantages over conventional biomaterials. Nanocrystalline materials are often produced by ball milling of coarse grained polycrystalline materials. Ball milling processing technique involves cold welding, fracturing and rewelding of powder particles, which has become an established technique for oxide dispersion strengthened materials and variety of metastable phases . Under equilibrium condition, the crystal structure of Ti metal is hcp (hexagonal close packed)--a phase or bcc (body centre cubic) b phase. It has been reported that hcp titanium can be transferred into fcc Ti . Chatterjee et al  concluded that ball milling of titanium sample for 10 h resulted in a mixture of nanocrystalline hcp and ~24 vol% fcc titanium. Lee et al fabricated nano-sized Ti[O.sub.2] powder via ethylene glycol method by employing water insoluble chemicals as a source of titanium ions. The Ti[O.sub.2] obtained was porous which was grounded to nanosized powder by planetary ball milling . Zhang et al  reported that titanium nitrides resulted by milling titanium powder with pyrazine, a ring type organic compound, in benzene solution up to 336h.
The bone bonding ability of a material is often evaluated in vitro by the formation of hydroxyapatite (HA) layer its surface in a simulated body fluid (SBF) with ion concentrations nearly equal to that of human blood plasma . The bone like apatite seems to activate signaling proteins and cells to start the cascade of events that result in bone formation (i.e the in vivo behaviour can be predicted by using in vitro tests such as immersion of synthetic materials into SBF solution). Pesakova et al., reported increased osteoblast adhesion in nano scale titanium observed that the osteoblast cells adhered specifically at particle boundaries for 1 h adhesion time . The microstructure of porous titanium scaffolds produced through a novel powder metallurgy process was also found to promote MC3T3-E1 pre-osteoblast proliferation and early differentiation compared to polished surfaces .
Nanostructured materials are now receiving much attention across many disciplines due to their novel and often improved properties over their conventional coarse grained counterparts. As there is a mismatch in modulus of elasticity between bone (5-30 GPa) and titanium metal (105 GPa), a porous structure would narrow the difference in the modulus. In this paper, pure titanium was ball milled to produce nanocrystalline particles without any contamination and was well characterized using XRD, SEM, TEM and mercury intrusion porosimetry (MIP) techniques. The ball milled powder were then compacted, sintered and subjected to in vitro bioactivity and osteoblast cell culture studies.
Materials and Methods
High purity titanium (99.7% Ti supplied by MIDHANI, Hyderabad) powder with initial size ~1 mm was used for ball milling. The chemical composition of the raw material is shown in Table 1. The powder was ground in a tungsten carbide closed vial with 10 mm diameter balls. Ethanol was used as the wetting medium. The powder to ball mass ratio was about 1:20 and the rotation speed was maintained at 200 rpm. The milling operation was performed during a cycle of 45 min on and 15 min off for cooling. Milling was done for different time intervals (4, 8, 12, 16, 20 and 24 h). The crystal structure of the as milled powder was characterized by X-ray powder diffraction (XRD) analysis with Bruker Discover D8 diffractometer using Cu [K.sub.a] radiation. The X-ray diffraction patterns (0.02^/ step from 30 to 80") were obtained for samples milled at various time intervals. Scanning electron microscopy (FEI Quanta 200, Holland) observations were performed to study the morphology of the milled powder. EDAX was also done in order to assess any phase or contamination occurring during ball milling. The ball milled nanoparticles were dispersed in ethanol using ultrasonicator and its morphology was observed in Philips CM12 transmission electron microscopy (TEM). The milled samples were compacted and sintered in a vacuum sealed quartz tube at 900 [degrees]C for 2 h. The pore size of the 20 h BM sample was obtained by using a mercury porosimetry (Micromeritics, AutoPore IV1.08). The sintered compacts were then immersed in simulated body fluid (SBF) for up to 4 weeks to access its bioactivity. SBF is prepared by dissolving reagent grade NaCl, KCl, NaHC[O.sub.3], MgS[O.sub.4].12[H.sub.2]O, Ca[Cl.sub.2] and K[H.sub.2]P[O.sub.4] into distilled water and buffered at pH = 7.3 with tris-hydroxymethyl aminomethane (TRIS) and HCl at 37 [degrees]C . After immersing in SBF for 2 and 4 weeks the samples were washed in distilled water, dried and its bioactivity is observed using SEM. EDAX was done to quantify the formation of HA. Apatite formed samples after immersion in SBF were also confirmed by XRD analysis.
For cell adhesion studies, the samples were steam sterilized for 3 h. It is then conditioned with sufficient volume of media. Human osteoblast cells were used to adhere on the surface of processed samples. The osteoblast cells (5000 cells/[cm.sup.2] surface area) were seeded on the surface of the samples and then sufficient media was added along the sides of the wall so that cell suspension will be on the top of the material. The culture plate was then incubated for 30-45 min in C[O.sub.2] incubator. Sufficient amount of media was again added and then incubated for 2 and 4 days. The samples were then removed and rinsed with phosphate buffer (PB) solution [Solution A--0.2M [Na.sub.2]HP[O.sub.4], Solution B--0.2M Na[H.sub.2]P[O.sub.4]; Mix 36 ml of solution A, 14 ml of solution B and 50 ml of deionised water]. It was then fixed in 2.5 % glutaraldehyde (Make 2.5 ml of 25 % glutaraldehyde to 25 ml using phosphate buffer) and rinsed with PB solution (5 times, 5 min each). After rinsing, the samples were dehydrated in different grades of alchohol.
The samples were then immersed in isoamylacetate for 2 min and dried to critical point in a critical point drier. The dried samples were gold sputter coated and observed in SEM.
Results and Discussions
The XRD data of ball milled titanium at various time intervals is shown in Fig. 1. It is found that the diffraction peaks became broader with increase in milling time, indicating the reduction in crystallite size of titanium. The powder particles tend to get cold welded to each other, especially if they are ductile, due to heavy plastic deformation experienced and then fractures during milling. During the initial stage of ball milling, cold welding is predominant than fracturing as the material is ductile. There should be a balance maintained between cold welding and fracturing of particles. Hence ethanol is added to the vials which act as surface active agents, adsorbs on the surface of the powder particles and minimizes cold welding between powder particles and there by inhibits agglomeration. It also acts as a coolant and avoids excessive heat formation during ball milling. After 24 h of ball milling, titanium oxide peaks were observed as shown in Fig. 1, At 24 h the titanium peaks were broadened and the oxide content increased with increase in milling time. This may be due nano sized crystallite having more surface area and entrapment of atmospheric air into the vial during sample loading. Nitrogen and oxygen are the two major compositions of atmospheric air which adsorbs on the newly created surfaces due to repeated fracture and cold welding during ball milling and diffuse into powder matrices through defects such as grain boundaries and dislocations. However, the formation enthalpies for TiN and rultile Ti[O.sub.2] are -346.89 and -959.75 kJ [mol.sup.-1] respectively at standard conditions. Hence, Ti[O.sub.2] should be easily formed than TiN at standard conditions .
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The morphologies for the samples milled at different time intervals were observed by SEM (Fig. 2). The particle size for the sample milled at 24 h was much smaller than that milled at different time intervals. This is because during ball milling a large amount of new surfaces through repeated fracture and cold welding occurs. As a result of heavy plastic deformation, work hardening takes place and the particles become less ductile and fracture easily at higher milling time. The EDAX at various milled time intervals is shown in Fig. 3 with a typical EDAX for 20 h BM sample. It is found that the oxide content increased with increase in milling time. The diffusion of oxygen into titanium matrix results in Ti[O.sub.2], a brittle phase which can be fractured easily. However, Ti[O.sub.2] is also bioactive and small amounts occurring during ball milling is unavoidable .
The morphologies of the milled samples were also observed by TEM. Fig. 4a & Fig. 4b shows the bright field and dark field images of 20 h ball milled samples. It was observed that the milled crystallites were agglomerated. This may be due to high surface energy of ball milled powder which tends to agglomerate the nano crystallites. The dark field image of the milled samples shows white spots in the agglomerated titanium crystallites, which indicates the presence of nano sized crystallites. The TEM micrograph for 24 h ball milled sample is shown in Fig. 4c. Selected area electron diffraction (SAED) pattern revealed that the diffraction spots were diffused to form a ring pattern which confirms the presence of nano sized crystallite.
The bioactivity of sintered compacts immersed in SBF is shown in Fig. 5. It was observed that the apatite formed on the surface of ball milled sintered pellets showed dense and homogenous coatings than in bulk titanium sample (Fig. 5e). This apatite forming process can be interpreted in terms of the surface charge. The treated titanium metal is initially negatively charged, and hence combines with positively charged calcium ions in SBF to form a calcium titanate. As the calcium ions are accumulated, the surface is positively charged and hence combines with negatively charged phosphate ions to form an amorphous calcium phosphate with low Ca/P ratio (~1.48). This phase is metastable and hence eventually transforms into crystalline bone-like apatite with Ca/P ratio almost similar to that of bone (~1.66). The apatite globules were much larger and densely packed when compared to all other conditions. The EDAX spectrum for 20 h ball milled sample immersed in SBF for 4 weeks is shown in Fig. 5d. Intensity of titanium peaks were less when compared to calcium and phosphate peaks. The atomic weight % of Ca/P ratio was found to be 1.66 which is close to the composition of bone.
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Porosity in the sintered samples also plays important role in enhancement of bioactivity. It has been reported that bone is a complex structure with hard cortical bone at the outer surface. The porosity varies from its outer region towards the core. Hence it is very difficult to control the porosity of artificial bone that allows imitating real bone so that the human body will accept and allow growth of tissues. From the mercury porosimetry, the interstitial porosity was found to be 48 % using Mayer-Stowe mercury breakthrough pressure. The interstitial pressure is the pressure required to penetrate a bed of packed spheres and the subsequent filling of the interstitial void . The average pore diameter was found to be 30 mm. As porous structure have more surface area than bulk samples, apatite precipitation was found in pore areas of the titanium pellets, but not on the dense ones, suggesting that porosity was necessary for inducing more apatite nucleation. The XRD data of sintered compacts immersed in SBF for 1, 2 and 4 weeks (Fig. 6) showed peaks at 26" and 32" at 2e which confirms the presence of apatite (JCPDS No. 9-432). The increase in the intensity of the HA peaks relative to titanium peaks of the 20 h BM sample after immersion in SBF for 4 weeks suggests an increase in the thickness of the apatite layer formed.
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The SEM images of cell adhesion test using human osteoblast for 2 and 4 days is shown in Fig. 7 and Fig. 8 respectively. After 4 days of culture, there were significantly more attached cells on the BM substrates than on the cover slip and bulk cpTi. The morphology of spread cells were classified into four stages according to Rajaraman et al. : stage 1, cells with a few filopodia in contact with the surface; stage 2, cells exhibit centrifugal growth of filopodia; stage 3, cells exhibiting cytoplasmic webbing; and stage 4, cells being fully spread and round or polygonal in shape. It was observed that after 4 days of incubation, the osteoblast cells exhibited advanced stages of spreading (i.e. stage 3 and stage 4) for the BM samples. On the other hand, few osteoblast cells were adhered on cover slip and bulk titanium samples when compared to porous titanium. Fig. 7(d) shows osteoblast cells exhibiting filopodia on BM samples after 2 days of incubation. Fig 8(d) shows magnified image of a single osteoblast cell incubated for 4 days. The trypsinized cells were spherical to ovoid in shape with 'bleb' like vesicles on the surface. These results indicate that a porous structure further enhances the osteoconductive properties of microrough surface by accelerating early osteoblast cell response. Thus the use of BM processed porous Ti substrate is an effective approach for accelerating the rate of osseointegration of porous Ti implants not only by promoting rapid attachment and spreading of osteoblasts on their surfaces but also by accelerating subsequent osteoblast differentiation.
The ball milling of high purity titanium in a closed vial formed nanocrystalline powder without any contamination. The sintered pellets of the milled powder exhibited dense and homogenous apatite layer than bulk titanium when immersed in SBF for 4 weeks. The formation of apatite on ball milled samples was enhanced due to the presence of porous structure. The morphology of the apatite was globular and its Ca/P ratio from EDAX was found to be 1.66 which is close to that of bone mineral phase. The osteoblast cell adhesion for 4 days indicated that the porous cpTi promotes better cell proliferation and differentiation.
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A. Thirugnanam, T. S. Sampath Kumar * and Uday Chakkingal
Department of Metallurgical and Materials Engineering, Indian Institute of Technology Madras, Chennai 600036, India.
* Corresponding author: T. S. Sampath Kumar (email@example.com)
Received 22 June 2010; Accepted 22 July 2010
Table 1: Chemical composition of titanium used for ball milling Elements Ti Fe Cl N O Si Ni % Composition 99.74 0.01 0.076 0.004 0.027 0.002 0.009 Elements C Others % Composition 0.004 < 0.03
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|Author:||Thirugnanam, A.; Sampath Kumar, T.S.; Chakkingal, Uday|
|Publication:||Trends in Biomaterials and Artificial Organs|
|Date:||Oct 1, 2010|
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