Hideo Kiba et al. : Bone Ingrowth into Porous HAp Journal of Hard Tissue Biology 21[3] (2012) p307-314 © 2012 The Hard Tissue Biology Network Association Printed in Japan, All rights reserved. CODEN-JHTBFF, ISSN 1341-7649 Original Bone Ingrowth into the Parallel Cylindrical Tubes with Different Sizes of Porous Hydroxyapatite Implanted into the Rabbits Hideo Kiba1), Noboru Kuboyama2), Ryoichiro Uchida3), Tsutomu Ishizaki4) and Norihiro Nishiyama3) 1)Department of Oral Pathology, Nihon University School of Dentistry at Matsudo, Chiba, Japan 2)Department of Pharmacology, Nihon University School of Dentistry at Matsudo, Chiba, Japan 3)Department of Dental Biomaterials, Nihon University School of Dentistry at Matsudo, Chiba, Japan 4)SANGI Co. Ltd., 2745-1 Fudoinno, Kasukabe, Saitama, Japan (Accepted for publication, May 4, 2012) Abstract : In this study, effects from pore size of the tube on the bone ingrowth and on the calcification of new bone permeated into the pore tube were examined through implantation of porous hydroxyapatite (HAp) in the lateral epicondyle in rabbits.The bone integration speed was strongly dependent on the pore size of the porous HAp. The 150-µm HAp provided a faster bone integration speed into the cylindrical tube than the 375-µm. However, when the pore size of the cylindrical tube in porous HAp was reduced to 20 µm, the bone integration speed into the cylindrical tube became slow. However, when the implanted period was prolonged to 8 weeks, the bone contact ratio into the 20-µm cylindrical tube dramatically increased and the bone density reached to74.0%. However, most new bones were remained as an osteoid bone. In contrast, the new bones formed inside of the 150-µm and 375-µm sized cylindrical tubes were calcified within the tube. It was concluded that the osteointegration into for the porous HAps and the calcification of osteoid formed were different with the pore size of the cylindrical tube. The optimum size of cylindrical tube was 150 µm. Key words: Porous hydroxyapatite, Osteoconduction, Implantation, Calcification Introduction between adjacent pores. It is well understood that hydroxyapatite (HAp) has a high Chang et al 2)designed a porous HAp with parallel cylindrical biocompatibility and bioaffinity to the hard tissues, since its pores of various sizes (50, 100, 300, 500 µm) without components are almost the same as that of the host bone 1-13). interconnecting fenestration between adjacent pores, and examined Porous HAp is considered a better substitute than dense HAp, the effect of pore size on the osteoconduction. They concluded since the wider the surface of the porous HAp becomes, the that the φ50-µm sized pore was enough for osteoconduction, and probability of the osteoconduction to the porous HAp becomes the φ300-µm sized pore was optimum. However, the effect from higher. Many researchers had designed the porous HAp with the the pore size of the cylindrical tube on the osteoconduction rate different size and geometry of the porosities by using coral, of the osteoid bone into the tube and the pore size effect on the naphthalene and polymer bead 4, 14-16). remodeling of the osteoid bone formed onto the inner wall of the Hubert et al 17) proposed the φ100 µm sized pore as the smallest tube were not identified yet. pore that can be used for osteoconduction, and the φ150µm sized In this study, in order to clarify the penetration rate of new pore as the optimal pore. Flatley et al 18) reported that the φ500 bone into the tube with parallel cylindrical pores and to determine µm sized pore was the most compatible for osteoconduction. the proper pore size to induce the calcified bone formation of However, most of these studies were based on the use of implants new bone within the cylindrical tube, the effect of pore size on of random pore geometry with interconnecting fenestration the osteoconduction rate into the tube in the porous HAp was examined. We determined the contact ratio of the new bone onto Corresponding author: Dr. Hideo Kiba, Department of Oral Pathology, the inner wall of cylindrical tube and the bone density of remodeled Nihon University School of Dentistry at Matsudo, 2-870-1, Sakaecho- bone at the bottom, middle, top portion in the porous HAp Nishi, Matsudo, Chiba, 271-8587, Japan. Tel: +81 47 360 9334, Fax: +81 structure. The null hypothesis tested was that the pore size of the 47 360 9335, E-mail: [email protected] 307 J.Hard Tissue Biology Vol. 21(3):307-314, 2012 HA-fine HA- middle HA-large (Diameter: 20 µm) (Diameter: 150 µm) (Diameter: 375 µm) Figure 1. Stereomicroscopic views of the porous HAp with different sizes of the parallel cylindrical tubes. The porous HAp has parallel cylindrical tubes without interconnecting fenestration between the cylindrical tubes. The mean pore sizes of the cylindrical tubes were φ 20, φ 150, and φ 375 µm, respectively. cylindrical tube in the porous HAp has no effect on the penetration of the new bone and the calcified bone formation of the new bone within the cylindrical tube. Materials and Methods 1. Cylindrical type HAp The porous HAp, which has different sizes of cylindrical tube, was supplied by Sangi Co (Tokyo, Japan). The porous HAp has parallel cylindrical tubes without interconnecting fenestration between the cylindrical tubes as shown Figure 1. The mean pore sizes of the cylindrical tubes were φ20, φ150, and φ375 µm, respectively. The porosity in the porous HAp was approximately 25% for the 20-µm HAp, 23% for the 150-µm HAp and 30% for the 375-µm HAp. 2. Surgical model The twenty Japanese white male rabbits, whose weight was about 4.0 kg and whose age was more than 2 years old, were used. The animals were randomly divided into 4 groups for only bone defect group (control), 20-µm HAp group, 150-µm HAp group and 375-µm HAp group. The porous HAps were sterilized by autoclaving and were then immersed in sterile saline immediately until the porous HAp was implanted. After anesthetized by using isoflurane nitrous oxide, a 2 cm Figure 2. Schema of implanted porous HAp into the lateral of the longitudinal incision was made on the lateral of the femur of the femur of the rabbit. The bone contact ratio and bone density of newly formed bone which penetrated along to the inner wall of rabbit and the lateral epicondyle was exposed. The periosteum the cylindrical tube of the porous HAp was determined at the top, was retracted, and a pilot hole was prepared by a round bur with middle and bottom portion in the porous HAp structure. The ratio dental hand engine. The hole was gradually widened by fisher of the calcified bone was also determined at the top, middle and bottom portion in the porous HAp structure.The square (400×400 burs with different diameters using a low rotational drilling speed µm) shows the area for analyzing the bone contact ratio, and the and internal cooling, until the diameter and the depth of the cavity bone density, and the ratio of the calcified bone. The position of became φ3.2 mm and 2.8 mm, respectively. The preparation of the square shows the analyzing position at the bottom, middle, and top portion. bone defect was done very gently using surgical technique. After the porous HAp, with a diameter of φ3.0 mm and a height of 2.7 mm, was implanted into the bone defect as shown in Figure 2, the soft tissues were closed in separate layers using resorbable sutures. 308 Hideo Kiba et al. : Bone Ingrowth into Porous HAp Figure 3. Light microscopic views of cross-section of the porous HAp, with different poresized of the cylindrical tube, implanted for 2 and 8 weeks (Azan stain, ×20). The upper views (a, b, c) were 2 weeks after implantation of the porous HAp, and the lower views (d, e, f) were 8 weeks after implantation. The views “a” and “d” were obtained from the 375-µm HAp, the views “b” and “e” were obtained from the 150-µm HAp, and the views “c” and “f” were obtained from the 20-µm HApAt 2 weeks after implantation, the new bone formation along to the inner wall of cylindrical tube in the porous HAp structure was observed at the bottom and middle portion in the porous HAp structure, when the 375-µm HAp (a) and the 150-µm HAp (b) was implanted. The newly formed bone was assigned to an un- calcificated osteoid tissue, because of the new bone being stained to blue by Azan staining. When the implantation period of the porous HAp was increased to 8 weeks, the new bone, which had formed at the bottom and middle portion of the inner tube for the 375-µm HAp (d) and the 150-µm HAp (e), was stained to red by the Azan stain. The calcified bone was formed by remodeling of the osteoid bone. Conversely, when the 20-µm HAp was implanted for 8 weeks, the new bone was not calcified and remained as an osteoid bone. At 2 weeks or 8 weeks after implantation, the rabbits were the cylindrical tube at the bottom, middle and top portion in the sacrificed by anesthetic overdose. The porous HAp with femur porous HAp structure in the porous HAp was determined by epicondyle bone marrow and muscle was removed. dividing the length of the inner wall where new bone had contacted, The rabbits were treated and kept according to the Japanese by the whole length of the inner wall occupied in an area of 1600 Laboratory Regulations. The study was approved by the Animal µm2 (400x400 µm) as shown in Figure 2.