Received date: 14 January 2011; Accepted date: 03 February 2011
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glass; apatite; wollastonite; osteoconduction; vivo
Glass-ceramics that contain apatite and wollastonite (GCAW) have been widely used in clinical applications because of their high bioactivity . In 2004, Ohsawa et al. described the excellent osteoconductive properties and material absorption abilities of commercially available GC-AW porous bodies . This study aims to determine the influence of the porous structure of GC-AW by using GC-AW with 5 different pore sizes.
Preparation of materials
Porous GC-AWwas prepared as follows. First, glass powder composed of 4.6% MgO, 44.7% CaO, 34.0% SiO2, 16.2% P2O5, and 0.5% CaF was prepared. The glass powder and polymeric beads were mixed with polyvinyl-alcohol solution, solidified, and dried at 60 °C. Two types of polymeric beads were used as spacer particles, namely, polymethylmethacrylate beads with a particle size of 300 μm and expanded polystyrene beads with a particle size of 1000 μm. The green compacts were heated to 580 °C to remove the polymeric beads and polyvinyl-alcohol and then sintered at 1050 °C in air. In order to change the microstructure of the porous bodies, they were heated at 2 different rates (G: 0.1 °C/min, S: 5 °C/min) in the temperature range of 580 °C to 800 °C, which includes the glass transition temperature. We designated the resultant 4 materials on the basis of the size of the spacer particles and heating rates as 1000G, 1000S, 300G, and 300S. The sintered porous bodies were cut into ø6 × 15 mmrods. Additionally, conventional GC-AW with a porosity of 70% and mean pore size of 200 μm was prepared (200S) . The characteristics of the 5 types of implants are provided in Table 1.
|Interconnecting pore size (μm)||175||175||20||20||50–80|
Table 1: Average pore diameter, porosity, and presence of microporosity.
Assessment of the morphology of porous GC-AW
The pore structure of the porous GC-AW was examined using a micro focus X-ray computed tomography system (micro-CT; SMX-100CT-SV3, Shimadzu Co., Japan). Distribution of interpore connections was determined on the basis of the penetration of Hg in an evacuated porosimeter (AutoPore IV9520; Shimadzu Co., Ltd., Kyoto, Japan).
To observe the inner pores, fractured porous AW was analyzed using scanning electron microscopy (SEM; S-4700; Hitachi Ltd., Tokyo, Japan).
To evaluate the osteoconductive ability in vivo, these samples were implanted into the femur metaphyses of 50 mature male Japanese white rabbits weighing 3.0–3.5 kg. The rabbits were sacrificed at 3, 6, and 12 weeks after implantation. This animal study was approved by the Animal Research Committee, Graduate School of Medicine, Kyoto University, Japan.
After the animals were sacrificed, the implants were removed, fixed in 10% phosphate-buffered formalin, and individually dehydrated in serial concentrations of ethanol. The specimens were then embedded in polyester resin, cut into sections of 80 μm thickness, and stained with Stevenel’s blue and Van Gieson’s picrofuchsin. A thorough microscopic analysis of the histological slides was performed using transmitted light microscopy.
To evaluate the bone ingrowth speed, we measured the depth of the new bone from the periphery of the implant. The rate of the bone ingrowth area (%) was measured on a personal computer using Adobe Photoshop CS3 and ImageJ (NIH). The rate of the bone ingrowth area was defined as the fraction of the bone area within the porous area available for bone ingrowth. Two sections surrounded by cancelous bone of either the medial or lateral condyles were examined in the case of each implant. Thus, 8 slices were analyzed for each type of implant at each implantation time. The bone area in the material was measured at 6 and 12 weeks after implantation, and the depth of bone ingrowth within the material was measured at 3 weeks. The significance of the difference in the degree was analyzed by analysis of variance (ANOVA).
Analysis of structure and morphology
Micro-CT and 3D reconstruction image analyses revealed the 3D interconnectivity of the porous structures of all the materials. The pore diameter of the macroporous structures, the diameter of interpore connections, and the total porosity were determined by mercury porosimeter and micro-CT analyses (Table 1).
Micropores (pore size, around 1 μm) were identified in the SEM image (Figure 1). These findings were compatible with the findings of mercury porosimeter analysis.
Bone ingrowth speed
In the case of 1000G, 1000S, and 200S, the bone ingrowth extended almost to the central section of the material (2.96± 0.04, 2.92 ± 0.08, and 2.90 ± 0.06 mm, respectively) at 3 weeks after implantation. In the case of 300G and 300S, the bone ingrowth extended up to one-third of the circle (1.34± 0.09 and 1.24 ± 0.11 mm, respectively). The rate of bone ingrowth was higher when 1000G, 1000S, and 200S were used than when 300G and 300S were used (P <0.01).
Histological findings and analysis of bone ingrowth area
Microscopic analysis revealed that new bone penetration extended throughout the central zones of all materials at 6 weeks. Vascular networks were well established and bone marrow had penetrated throughout the central zone; furthermore, the bone had a more mature, organized, lamellar structure at 6 weeks (Figure 2).
The percentage of bone ingrowth area significantly increased in the case of 300G, 300S, and 200S at 6 and 12 weeks after implantation compared to that in the case of 1000G and 1000S (P <0.05) (Figure 3).
Absorption of all materials was clearly visible, especially in the case of 300G and 200S (Figure 2).
Porous bioactive ceramics and glass implants have been widely used in the clinical setting as a bone substitute. In the case of these materials, 2 important factors affect osteoconduction, namely, their physicochemical characteristics (e.g., hydroxyapatite, glass, and TCP) and porous topography.
Porous topography includes the macropore, interconnective pore, and micropore sizes as well as pore interconnectivity. In scientific literature, determination of the optimum pore size has been the topic of numerous studies. Generally, a macropore size of 50–800 μm and an interconnection pore size of 50–100 μm are considered adequate . It is still controversial whether or not micropores are essential for osteoconduction. Our results suggest that GC-AW with a pore size of 800 μm is unsuitable for bone growth, and rapid osteoconduction can be achieved at interconnecting pore sizes exceeding 50 μm. These results are consistent with those of other reports. The interconnecting pore sizes in the case of 300G and 300S are approximately 20 μm, and this size maybe too small for osteoconduction; however, GC-AW is a resorptive material, and its interconnecting pores may expand with the implantation period. As for the micropores, they may not be important for osteoconduction in the case of highly bioactive materials like GC-AW.
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