Study of Apatite Deposition in a Simulated Body Fluid Immersion Experiment

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1 J Oral Tissue Engin 2016;14(1):9-14 ORIGINAL ARTICLE Study of Apatite Deposition in a Simulated Body Fluid Immersion Experiment Hajime SUZUKI 1,2, Ryo YAGI 3, Takuya WAKI 3, Takeshi WADA 1,4, Chikahiro OHKUBO 3, and Tohru HAYAKAWA 1 1 Department of Dental Engineering, Tsurumi University School of Dental Medicine, Yokohama, Japan 2 Institute of Clinical Materials, Osaka, Japan 3 Department of Removable Prosthodontics, Tsurumi University School of Dental Medicine, Yokohama, Japan 4 Institute of Implant Reconstructive Dentistry, Tokyo, Japan SYNOPSIS The present study evaluated the influence of two different placement orientations of biomaterials on apatite deposition behaviors during immersion in simulated body fluid (SBF). Titanium (Ti) and thin carbonate-containing hydroxyapatite coated Ti (CA/Ti) were used as biomaterials, and Hank s balanced salt solution was employed as an SBF. Ti and CA/Ti disks were placed either horizontally or vertically. For the horizontal placement, the disks were placed directly on the bottom of the container. For the vertical placement, the disks were hung from a nylon wire. The medium and container were replaced every day. After immersion for 3, 7, and 14 days, the surface appearances of the horizontal or vertical sides of the Ti and CA/Ti disks were observed using a scanning electron microscope. Ti disks showed apatite deposition after 3 days of immersion with the horizontal placement, but few disks showed this with the vertical placement. The Ti surface of the horizontal side was completely covered with deposited apatite. Even after 7 and 14 days, the whole Ti surface of the vertical disk was not completely covered with deposited apatite. For CA/Ti, apatite deposition was clearly recognized after 3 days of immersion on the surfaces of not only the horizontal placement but also the vertical placement. Piled up apatite globules were observed on the horizontal surface, but no distinct piled up globules were observed on the vertical surface after 7 and 14 days of immersion. In conclusion, it revealed that apatite deposition and apatite crystal growth were influenced by the placement orientation of sample disks in SBF immersion experiments. Key words: simulated body fluid, Hank s balanced salt solution, apatite deposition, titanium, thin apatite coating INTRODUCTION Various kinds of bone substitute materials have been developed and evaluated for their bone biocompatibility. The evaluation of biocompatibility is mostly based on cell assays or animal experiments. Conversely, Kokubo 1 proposed that the formation of bone-like apatite on 9

2 Suzuki et al., Apatite Deposition in Simulated Body Fluid a material surface is essential for the bonding of artificial materials to living bone, and in vivo apatite formation can be reproduced in a simulated body fluid (SBF). The ion concentrations of SBF are nearly equal to those in human blood plasma. Kokubo and his colleague have reported numerous studies on the effectiveness of SBF immersion experiments, and they maintain that in vivo bioactivity, namely osteoconductivity, of biomaterials is precisely mirrored by their in vitro apatite forming ability in SBF 2-7. Using SBF immersion experiments can reduce the number of animals used and the duration of animal experiments. In addition to SBF proposed by Kokubo, Hanawa et al used Hank s balanced salt solution (HBSS) without organic species at ph 7.4 as SBF and found that apatite formed on a titanium (Ti) surface after immersion in HBSS, thereby indicating the efficacy of HBSS as an alternative to SBF. Hayakawa and colleagues examined apatite deposition on biomaterials, such as thin-apatitecoated Ti or Ti fiber, poly(lactide-coglycolide) composite, partially stabilized zirconia, and DNA-coated Ti, after the immersion in SBF and also found that better apatite deposition on the biomaterials in HBSS corresponded with better in vivo bone formation In living tissue, apatite deposition occurs from various directions. It has been speculated that the surface direction of biomaterials in SBF will influence apatite deposition on the material surface. In the present study, the influence of two different placement directions of biomaterials on apatite deposition behaviors in SBF immersion experiments was evaluated. Ti and thin apatite-coated Ti disks were placed in HBSS horizontally and vertically, and the differences in apatite deposition behaviors were monitored. MATERIALS AND METHODS Commercially pure Ti disks (Grade 2, Furuuchi Chemical Corp., Tokyo, Japan) with a diameter of 1.5 mm and thickness of 1.0 mm were used. A thin apatite coating was applied using a molecular precursor method 14,17. Briefly, 25 L of molecular precursor solution, which is an ethanol solution of Ca-ethylenediaminetetraacetic acid (EDTA) complex and dibutylammonium diphosphate salt with Ca/P ratio of 1.67, was applied to Ti disks, which were then spin-coated at 500 rpm for 5 s and 2000 rpm for 30 s using a spin coater (1H-D7, MIKASA, Tokyo, Japan). The precursor solution-covered Ti disks were heated at 600 C for 2 h using a tubular furnace (EPKPO12-K, ISUZU, Niigata, Japan) in a stream of oxygen gas introduced at a rate of 100 ml/min. A thin carbonate-containing hydroxyapatite (CA) coated Ti (CA/Ti) disk was obtained. The ion concentrations of HBSS are listed in Table 1. Ti and CA/Ti disks were immersed in 20 ml HBSS with an adjusted ph to 7.4 at 37 C in a polypropylene container. Two different placements, i.e. horizontal and vertical, of the Table 1 Concentrations of electrolytes in HBSS Ion Na + K + Mg 2+ Ca 2+ Cl - 2- HPO 4 SO 4 2- HCO 3 - Concentration (mmol/l)

3 J Oral Tissue Engin 2016;14(1):9-14 disks were evaluated, as shown in Fig. 1. For the horizontal placement, disk was directly placed on the bottom of the container. For the vertical placement, the disk was hung from a nylon wire. The nylon wire was attached to disk using a plastic clip. The medium and containers were replaced every day to expose the disks to fresh medium. After immersion for 3, 7, and 14 days, HBSS on the disks was removed using soft paper, and the disks were immediately dried in a desiccator. The surface appearances of the horizontal or vertical sides of the Ti and CA/Ti disks after immersion in HBSS were observed using a scanning electron microscope (SEM; JSM-5600LV, JOEL, Tokyo, Japan) at an accelerating voltage of 15 kv. Materials were sputter-coated with gold prior to being examined. RESULT SEM pictures of the surface appearances of the horizontal or vertical side of the Ti and CA/Ti disks after immersion in HBSS are shown in Figs In Fig. 2, a Ti disk placed horizontally showed apatite deposition after 3 days of immersion in HBSS. The surface of the horizontal side was completely covered with deposited apatite. After 7 and 14 days, more apatite deposition was observed on the Ti surface. In contrast, for the vertical placement, very little apatite deposition was observed on the Ti surface after 3 days of immersion, as shown in Fig. 3. After 7 days of immersion in HBSS, apatite deposition was clearly observed. However, the whole Ti surface of the vertical side was not completely covered with deposited apatite. After 14 days of immersion in HBSS, a greater amount of apatite deposition was identified, but detailed observations also revealed that whole Ti surface was not completely covered with deposited apatite. Fig. 1 Schematic drawing of the horizontal and vertical placement of sample disks in HBSS. Fig. 2 SEM pictures of the surface appearance of Ti disks after horizontal immersion in 11

4 Suzuki et al., Apatite Deposition in Simulated Body Fluid Fig. 3 SEM pictures of the surface appearance of Ti disks after vertical immersion in Fig. 4 SEM pictures of the surface appearance of CA/Ti disks after horizontal immersion in Fig. 5 SEM pictures of the surface appearance of CA/Ti disks after vertical immersion in 12

5 J Oral Tissue Engin 2016;14(1):9-14 For CA/Ti disks, apatite deposition was clearly visible after 3 days of immersion in HBSS on the surfaces of not only the horizontally placed disks but also on the vertically placed disks, as shown in Figs. 4 and 5, respectively. However, the deposition morphologies of apatite were different between the horizontal and vertical placements after 7 and 14 days of immersion in HBSS. Piled up apatite globules were observed on the surface of the horizontal disk (Fig. 4), but no distinct piled up globules were observed on the surface of the vertical disk (Fig. 5). DISCUSSION In the present study, we evaluated the apatite deposition behaviors on Ti and CA/Ti surfaces for horizontally and vertically oriented disks in HBSS. It was revealed that the different placements caused the different behaviors of apatite deposition. No apatite deposition was observed on the Ti surface for vertical disks after 3 days of immersion in HBSS. For CA/Ti disks, apatite deposition was observed on the vertical disk after 3 days of immersion, but a different deposition behavior was observed after 7 and 14 days of immersion. Nucleation and deposited crystal growth governed the apatite deposition behavior in SBF immersion experiments. It was presumed that no nucleation occurred on the vertical Ti surface after 3 days of immersion. Between 3 and 7 days of immersion, nucleation and deposited crystal growth occurred on some of the vertical Ti surface, but not over the whole surface. Even after 14 days of immersion, the whole Ti surface was not completely covered with apatite crystals. This was due to insufficient nucleation on the vertical Ti surface. Conversely, apatite deposition on the vertical CA/Ti surface was observed after 3 days of immersion. This revealed that the CA surface induced the nucleation of apatite crystals during HBSS immersion. This nucleation performance corresponded with the excellent osteoconductivity of the CA coating 11,12,14. However, growth of deposited apatite crystals was different between the horizontal and vertical disks. It was presumed that sufficient apatite crystal growth in the horizontal direction was difficult for the vertical placement. Thus, the present study suggested that in vivo apatite formation on the Ti surface may be influenced by three-dimensional directions and heterogeneous apatite deposition or crystal growth may have occurred. The crystal characterization of deposited apatite and/or analysis of the amounts of deposited apatite need further investigation. Moreover, living body fluid in vivo has a flow system, but the present HBSS did not flow. Apatite crystallization on Ti in flowing Kokubo s SBF solution has been evaluted 18, and the influence of HBSS flow on apatite deposition should be further investigated. For thin apatite coating, a molecular precursor method was employed 14,17). The advantage of the molecular precursor method is its simplicity and also that the CA coating can be deposited onto substrates of any shape. It was possible for uniform CA coating on the inside of a three-dimensional Ti fiber structure using the molecular precursor method 19. In conclusion, the present study revealed that apatite deposition and apatite crystal growth were influenced by the orientation of the sample material during immersion in SBF. In vivo mineralization is a complex system, and more detailed studies for the simulation of apatite deposition are needed. 13

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