Long-term observation of subcutaneous tissue reaction to synthetic auditory ossicle (Bioceram®) in rats Chun-Sheng Zhua, Katsuichiro Ohsakia, Kunio Iib,Qing Yeb, Yen Hai Trana, Yasuo Ohbac, and Keiji Moriyamac aDivision of Clinical Otology, University Hospital, bFirst Department of Pathology, The University of Tokushima School of Medicine, Tokushima, Japan;and cDepartment of Orthodontics, The University of Tokushima School of Dentistry, Tokushima, Japan Abstract:To evaluate biocompatibility to tissue in long-term implantation, Bioceram® discs made of aluminum oxide (Al2O3) were implanted subcutaneously within the interscapular region of 64 rats for six to 20 months. Histological sections stained with haematoxylin and eosin (H&E) and the surface of the implant material were observed using light micros-copy. Different cell types and the thickness of fibrous capsules surrounding the implants were examined quantitatively by light microscopy. Small numbers of macrophages (2.8±0.7%) and lymphocytes (2.7±0.9%) were observed at six months after implantation, gradually decreasing to zero at 16, 18 and 20 months. Neither neutrophils nor foreign body giant cells were seen in any specimens. The thickness of fibrous capsules surrounding the implants was closely related to the shape of the implant, but there was no significant change between six and 20 months after implantation. No change in Bioceram® surfaces were observed under stereoscopic microscopy from six to20months after implantation. The study results indicate that Bioceram® is a satisfactory biocompatible material for reconstructive surgery from the viewpoint of long-term tissue response. Present results of experiments with Bioceram® are also compared to previous results with Apaceram® and different tissue responses of the two materials are discussed. J. Med. Invest. 46:97-103, 1999 Keywords:aluminum oxide, long-term implantation, subcutaneous tissue reaction, histology, rats INTRODUCTION The synthetic auditory ossicle (Bioceram®) com-posed of the bio-inert ceramic material aluminum oxide (Al2O3) is currently used widely in reconstruc-tive middle ear surgery. Bioceram®'s high biocom-patibility has been reported (1, 2) and our previous study showed a relative low inflammatory cell re-sponse to Bioceram® in the early stages after implan-tation (3). However, histological changes at the Bioceram®-tissue interface and surfaces change of implants during long-term implantation have received limited attention. In the present study, small and thin Bioceram® discs were implanted into the sub-cutaneous tissue of rats with the aim of investigating histological reactions between six and 20 months after implantation. The thickness of the fibrous cap-sules surrounding Bioceram® discs was examined quantitatively by light microscopy;surface changes in Bioceram® discs were examined by stereoscopic microscopy. The experimental results on Bioceram® were compared with the results of our previous study (4) using bioactive synthetic auditory ossicle (Apaceram®) made of hydroxyapatite [Ca10(PO4)6(OH)2]. MATERIAL AND METHODS Implant material Dense discs (diameter, 4mm;thickness, 1mm) of Bioceram® were prepared from commercially available synthetic auditory ossicle (Kyocera Co. Ltd, Kyoto, Japan). Before implantation, the discs were sterilized in an autoclave at 121°C for 30minutes. Animals and implantation Bioceram® discs were implanted subcutaneously within the interscapular region of 64 eight-week-old female SPF Wistar rats under general anesthesia (diethyl ether) in a sterile environment. Wounds were closed by suturing. Histological procedures and observation Rats were sacrificed quickly in groups of 8 at6, 8, 10, 12, 14, 16, 18 and 20 months after implantation by general anaesthesia using diethyl ether. The Bioceram® discs and surrounding tissue were re-moved as a single mass and immediately immersed in 10 per cent phosphate-buffered formalin for three days. The Bioceram® discs were carefully removed from the tissue mass under Stereoscopic microscopy to minimize damage to the tissue surrounding discs. Tissue surrounding the discs was dehydrated in an ethanol series. After being embedded in paraffin, 10 to 15 sections (6μm thick) from each specimen were stained with haematoxylin and eosin (H&E). Five randomly chosen sections per specimen were observed and photographed under light microscopy. Photographic slides were projected and cells were identified and counted. Each specimen had a total of between 202 and 607 cells, and percentages of various cellular components were calculated. One randomly chosen section per specimen was observed under light microscopy and the thickness of the fibrous capsule surrounding the Bioceram® disc was measured. Figure1a shows a Bioceram® disc divided into:1) flat portions (upper and lower portions), 2) lateral portions, and 3) ring portions (upper ring-shaped and lower ring-shaped portions). Figure1b shows the fibrous capsule attached to the disc surface. Photographic slides of the fibrous cap-sules were projected on a screen and the thickness of the capsules was measured using an objective micrometer at the same magnification as samples. Average thickness values for each group of 8 rats were calculated at flat portions, lateral portions and ring portions. Untreated Bioceram® disc and Bioceram® discs obtained from specimens were observed under stereoscopic microscopy. Statistical analyses were performed on a Macintosh Performa 588computer with the Excel 5.0 statistical program. Difference was calculated using Student's t-test (two-tailed) with a level of p<0.05 being accepted as significant. RESULTS Surface observation of Bioceram® discs Stereoscopic microscopy examination showed no surface changes in the Bioceram® discs from six to 20 months after implantation in comparison with untreated Bioceram® surfaces Histological observation General observation and cell distribution Fibrous capsules surrounding implant discs were seen in all sections between six and 20 months after implantation (Fig. 1b). Capsules were composed of macrophages, lymphocytes, fibroblasts and fibro-cytes, as well as collagen, and in some cases, cap-illaries. Macrophages, lymphocytes and fibroblasts were located close to the Bioceram® disc-tissue interfaces. Fibrocytes were located in the outer layers of the fibrous capsules. Cell population Table1 shows the cellular components surround-ing Bioceram® discs. The proportion of macrophages was 2.8±0.7% and the proportion of lymphocytes was 2.7±0.9% at six months after implantation (Fig. 2), gradually decreasing to 0.4±0.6% for macro-phages and 0.1±0.2% for lymphocytes at 14 months. At 16 months, macrophages and lymphocytes com-pletely disappeared (Fig. 3). Percentage of fibroblasts was 25.4±3.2% at six months, gradually decreasing to 1.8±1.7% at 20months. In contrast, fibrocytes increased from 66.7±2.4% at 6months to 97.3±2.2% at 20 months. The average±SD of absolute number of infil-trated cells in Apaceram® and Bioceram® is shown in Table2. Thickness of fibrous capsules surrounding Bioceram® discs Table3 shows the average thickness of fibrous capsules surrounding Bioceram® discs at 1) flat portions, 2) lateral portions, and 3) ring portions. In general, fibrous capsules were thickest at the flat portions and thinnest at the ring portions in every test period. Thickness of fibrous capsules at the flat portions tended to increase from 6months(95.1±16.5μm, n=16) to 20months (118.2±48.0μm, n=16) after implantation, but the difference was not significant. Thickness of fibrous capsules changed slightly at the lateral portions from 28.5±5.0μm to 39.2±11.1μm and at the ring portions from 8.2±1.3μm to 10.7±1.6μm in the experimen-tal period. DISCUSSION Accurate assessment of implant materials requires long-term investigation of the tissue reaction sur-rounding the implant material and analysis of the physico-chemical changes in the implanted material. Macrophage reaction to Bioceram® at six months and longer after implantation Clearly, the macrophage is the dominant cell type at the implant surface, playing a major role in cel-lular response and tissue reaction to the implant (5). Implant stability depends largely on the dynamic behavior of macrophages (6). Macrophages accu-mulating at the implant-tissue interface can produce various secretions including:1) chemotactic agents for other cells, 2) growth factors which stimulate production of collagen by fibroblasts, and 3) neutral proteases which may affect the implant surface. Therefore, population and activities of macrophages at the implant-tissue interface may reflect the bio-compatibility of a biomaterial (7, 8). In the present study, macrophages accounted for 2.8±0.7% at six months after implantation, gradually decreasing to0.4±0.6% at 14 months and completely disappearing at 16 months. So, from the viewpoint of cellular response, Bioceram® is probably a satisfactory bio-compatible material for long-term implantation. Fibrous capsules surrounding Bioceram® The thickness of fibrous capsules surrounding an implant is also an important indicator in evaluating the biocompatibility of artificial material (2, 8). In the present study on Bioceram®, the thickness of fibrous capsules from different regions of the same sample differed substantially. Fibrous capsules from flat portions of discs were much thicker than from other portions, and the ring portions were thinnest. These data agree with another reports (9), indi-cating that capsule formation is closely related to implant shape. Contact of disc surface with tissue is influenced physically and/or chemically by both area and shape (10). Different physical and/or chemical stimulation at the flat portions, lateral portions, and ring portions may cause varying thick-nesses of fibrous capsules. Future experiments must investigate precisely how the different portions of a synthetic prosthesis disc affect implant biocompat-ibility. The thickness value in the flat portions increased by 24.3% from six to 20 months, but there was no statistically significant difference. The thickness of fibrous capsules surrounding Bioceram® discs was stable at least between six and 20 months after implantation, and this is consistent with the report by Boutin et al. (11) Comparison of Bioceram® and Apaceram® Both Bioceram® and Apaceram® are popular ma-terials in middle ear reconstructive surgery. Tissue response to Apaceram® in long-term implantation was investigated in our previous study (4). The results of the present study on Bioceram® were compared with the previous results on Apaceram®, because while Apaceram® is a bioactive material, Bioceram® is regarded as a bioinert material. Appearance of macrophages and foreign body giant cells The number of macrophages surrounding Apaceram® was remarkably higher than the number of macro-phages surrounding Bioceram® (Fig. 4a). A small number of foreign body giant cells (0.5 - 0.8%) was found at Apaceram®-tissue interfaces between six to 20months after implantation (4). However, no foreign body giant cells were seen in any sections removed from specimens of Bioceram® between six and 20 months over the same period of time after im-plantation. Presumably, tissue reaction to Bioceram® from six to 20 months after implantation is milder than tissue reaction to Apaceram®. Differences in macrophage and foreign body giant cell responses to Bioceram® and Apaceram® may be explained by the different physico-chemical properties, biomechanical compatibility, surface tex-ture, and solubility of the two materials to tissue. Aluminum oxide, a material in the highest state of oxidation, is thermodynamically stable with an ionic structure that creates a hydrophilic surface with high wettability, possibly resulting in low tissue reaction (12). In vivo and in vitro experimen-tal studies show that aluminum oxide releases few ions into surrounding areas (13). In addition, low stimulus to macrophages is reported (14, 15). Hydroxyapatite is a calcium phosphate bioce-ramic, the major inorganic component of bone. Hydroxyapatite shows significant ion release in vitro and in vivo (16-18). Biodegradation (16), demin-eralization and remineralization of hydroxyapatite have been reported after implantation (17, 18). In the process of these changes, some particles and ions permeate surrounding tissue and stimulate inflammatory cells. Activated macrophages aggres-sively fuse to form foreign body giant cells with a few particles in the cytoplasm of macrophages and foreign body giant cells (4, 19). However, alumi-num oxide does not create such responses in the living body. Cellular response to aluminum oxide completely disappeared during the later stages of the present experiment. However, a small number of macrophages, lymphocytes and foreign body giant cells were continuously present at Apaceram®-tissue interfaces, even up to 20 months after implantation(4). Another possible explanation for the differences in macrophages and foreign body giant cell re-sponses in Bioceram® and Apaceram® is that the roughness of implant surfaces is associated with the appearance of macrophages and foreign body giant cells (2). The mechanism is unclear, but rough im-plant surfaces have resulted in significant increases in the proportion of surface covered by macro-phages and foreign body giant cells (20-22). In our studies, both the Bioceram® and Apaceram® discs had a smooth surface before implantation. How-ever, surfaces of Apaceram® discs became rough after implantation because of the above-mentioned physico-chemical changes (4, 17). In contrast, ex-amination by stereoscopic microscopy showed no change to the surface of Bioceram® discs. The rough surface of Apaceram® discs possibly caused macrophages and foreign body giant cells to be continuously present at the implant-tissue interfaces during long-term implantation. Fibroblasts and fibrocytes Fig. 4b shows that the fibroblast level surround-ing Apaceram® is higher than the fibroblast level surrounding Bioceram®. Statistical analyses showed no significant differences at 6, 8 and 10months, but there were significant differences from 12 to 20months after implantation (p<0.05). The population of fibrocytes surrounding Bioceram® was signifi-cantly higher than that surrounding Apaceram® at all experimental periods (p<0.01). Fibrocytes usually demonstrate mature fibrous connective tissue. Thus, Bioceram® seems to exhibit a satisfactory tissue-implant relation. Fibroblast ability is mainly controlled by macrophages in wound healing and cellular responses to long-term implants (6, 8). Therefore, different macrophage levels surrounding Bioceram® and Apaceram® may result in different fibroblastic and fibrocytic reactions. CONCLUSION The present study showed eventual disappear-ance of inflammatory cell response to Bioceram® and well-matured connective fibrous capsules sur-rounding Bioceram® discs from six to 20 months after implantation. These results suggest that, from the viewpoint of long-term tissue response, Bioceram® has satisfactory biocompatibility for use as an implant material for reconstructive surgery. ACKNOWLEDGEMENTS This study was supported in part by a Grant-in-Aid (No.10671594) for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. REFERENCES 1. Yamamoto E, Iwanaga M:Soft tissue reaction to ceramic ossicular replacement prosthesis. J Laryngol Otol101:897-904, 1987 2. Takeshita F, Morimoto K, Suetsugu T:Tissue reaction to alumina implants inserted into the tibiae of rats. J Biomed Mater Res 27:421-428, 1993 3. 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Address correspondence and reprint requests to Katsuichiro Ohsaki, M.D., Ph.D., Division of Clinical Otology, University Hospital, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima 770-8503, Japan and Fax:+81-88-633-7208.