A subcutaneous tissue reaction in the early stage to a synthetic auditory ossicle (Bioceram®) in rats

A subcutaneous tissue reaction in the early stage to a synthetic auditory ossicle (Bioceram®) in rats

Qing Ye^a, Katsuichiro Ohsaki^b, Kunio Ii^a, Dong-jun Li^b, Hiroshi Matsuoka^b,
Satoru Tenshin^c, and Teruko Yamamoto^d

^aFirst Department of Pathology, ^bDivision of Clinical Otology, University Hospital, The University of Tokushima School of Medicine, Tokushima, Japan;^cDepartment of Orthodontics, The University of Tokushima School of Dentistry, Tokushima, Japan;and ^dDepartment of Orthodontics, Okayama University Dental School Okayama, Japan

Abstract:The discs of synthetic auditory ossicle (Bioceram®), which are composed of aluminium oxide (Al2O3), were implanted subcutaneously in the interscapular region of 16 rats. The implanted specimens were removed at 1, 3, 7 and 14days after implantation. The decalcified 6μm thick sections were stained with H.E. and cell types around the implants were counted microscopically. We found that an acute inflammatory reaction occurred at one day, in which macrophages and neutrophiles predominated, and almost disappeared at about 7days after implantation. Fibrosis began to be observed at 3days. During this early stage, foreign body giant cells were found in only one specimen at 3days. These findings, in comparison with those in the controls, showed that the chemical irritation of Bioceram® to the subcutaneous tissue is slight, although the physical and/or chemical irritation of Bioceram® lasts continuously and induces fibrosis around the bioimplant. The results so far suggest that Bioceram® seems to be a satisfactorily biocompatible material, at least within the extent of 2weeks. J. Med. Invest. 44:173-177, 1998

Keywords:aluminum oxide ceramics;histology;cell count;biocompatible materials;rats

INTRODUCTION
Biomaterials are the building blocks of implant technology. Biomaterials used in otolaryngological surgery can be grouped into four categories, on the basis of their material science definition:metals, ceramics, polymers, and composites (1). There had been little study about the histopathological changes in the biomaterial-tissue interface during the early stage of implantation, we therefore decided to investigate the early response of soft tissue to implanted biomaterial. In this study, we chose Bioceram® which contains aluminum oxide ceramics, one of ceramics among those four categories mentioned above, as a experimental bioimplant. However if histologic evaluations of the soft tissue response to biomaterials were merely based on qualitative diagnosis or semiquantitative grading, there would be an inherent weakness due to the subjec-tivity of the observers. To overcome this drawback, a quantitative approach is preferable (2), so we implanted small, thin discs of Bioceram® subcutaneously in rats and observed the resulting histological reaction quantitatively by light microscopy from 24 hours to 2 weeks after implantation.

MATERIALS AND METHODS
Implant material characteristics : Dense discs (diameter, 4mm;thickness, 1mm) of Bioceram® (Al2O3) were prepared from commercially available synthetic auditory ossicle (Kyocera Co. Ltd., Kyoto). Before implantation, the discs were sterilized in an autoclave at 121°C for 30minutes.
Animals and operations:Twenty-four eight-week-old female SPF Wistar rats were divided into two groups. Bioceram® disc were implanted subcutaneously into the interscapular region of 16rats as a test group. Eight rats underwent the same operation but did not received any implants as a control group. Four rats from the test group and two from the control group were sacrificed at1, 3, 7or14days after implantation. All surgical procedures were carried out in sterile conditions under general anaesthesia using diethyl ether.
Section preparation and staining:The Bioceram® disc and its surrounding tissue were removed as a single mass and immersed immediately in 10 percent phosphate buffered formalin, then dehydrated in an ethanol series. Seeing that Bioceram® can not be decalcified, and in order to get thin and fine sections, we took Bioceram® discs out of soft tissue mass very carefully without any damage to tissues under microscope before the paraffin embedding procedure (Figure1). After being embedded in paraffin, the tissue mass was cut into sections 6μm thick. The sections were stained with haematoxylin and eosin.
Observation methods:The types and distribution of cells around the implants were photographed (colour slides) under a light microscope. The pictures were enlarged with a slide projector to identify and the different cells were counted. Over 200 cells were counted for each specimen and the percentages of the various component cells were calculated for each group.

RESULTS
The tissue around the implants had two main structures, a layer of acute inflammatory cells, such as neutrophils, macrophages and lymphocytes, and a surrounding layer of fibrous tissue composed of fibroblasts and fibrocytes, and in some cases capillaries. The average percentages of the component cells are shown in Table1.
Sections from specimens which were removed one day after implantation showed an acute inflammatory response around the implants (Figure2a, b). Cell counts revealed a predominance of macrophages (69.7%), followed by neutrophils (19.6%) and lymphocytes (8.7%). Neither fibroblast nor fibrocyte proliferation was observed in any of the specimens.
Specimens taken three days after implantation (Figure3) showed a sharp decrease in neutrophils to almost zero(0.4%), the components of macrophages and lymphocytes also decreased to 53.3% and 4.4% respectively. In this group, both fibroblasts and fibrocytes began to be observed (33.1% and 5.5%). 0.2% foreign body giant cells (FBGCs) were noticed in one specimen in this group (Figure4).
Sections from specimens which were removed seven days after implantation (Figure5) showed a decrease of macrophages to19.4%. We could not find any neutrophils. The implants were surrounded by a layer of fibroblasts(52.2%) and fibrocytes (23.6%).
Specimens obtained14days after implantation (Figure6) showed that macrophages still remained at a low level(3.0%). The implants were surrounded by a thin layer of fibroblasts (44.7%) and fibrocytes (48.2%), and only a few lymphocytes were still evident (1.2%).
Compared to the test groups, the controls showed the presence of macrophages (85.3%) one day after surgery, and these had completely disappeared by the seventh day after surgery. Neutrophils (9.2%) were evident only one day after surgery. Fibroblasts and fibrocytes increased gradually after three days. The microscopic changes evident in the controls were typical of those normally resulting from surgical intervention.

DISCUSSION
After implantation of a biomaterial, the responses that occur at the interface of the implant and in the sur-rounding environment are important in determining its biocompatibility. In our study, it revealved that there were two main structures, one was acute inflammatory cells, another one was fibrous tissue and in some cases capillaries, the results we obtained are the same as the finding which other authors described using other sorts of biomaterials such as hydroxyapatite [Ca10(PO4)6(OH)2] and bioceramics (3, 4, 5). As a kind of synthetic material, Bioceram® is a ceramics, which is made of aluminium oxide (Al2O3) (6). It is reported that Bioceram® has merits such as good stability (bioinert), affinity, anti-corrodibility (6). The biological response to an implant can be attributed to acute and chronic inflammatory changes, following the formation of a fibrous capsule, which occurs over a period of time (7). In the present study, the results acquired in the control group within two weeks were compatible with the normal healing of skin wounds. In the test group, the percentages of neutrophils and lymphocytes were approx-imately two and three-fold greater respectively than those in the control groups. The neutrophils had sharply decreased at three days after implantation, whereas the lymphocytes persisted at a lower level until day14after implantation. The peak of macrophages in both groups appeared at the same first day, and these cells had almost disappeared at two weeks after implantation, which was nearly one week later than in the controls. This must have been due to the presence of the implant, and indicated an attempt by the macrophages to induce healing around the bio-material like organ repair following injury (8, 9). There is no doubt that macrophages play a central role in wound repair and tissue reorganization (10). They have been shown to have the ability to control fibroblast activity, and thus indirectly the formation of collagen (8, 9, 2).
The stability of an implant depends largely on the behavior of cells at the material-tissue interface. Since macrophages are cells which are particularly relevant to material compatibility (10), their changes and activities around an implant in the period after implantation may reflect the biocompatibility material (2). Macrophage counts at two weeks after implantation were only three percent. Microscopic observation of the specimens after implantation also confirmed that the Bioceram® discs were encapsulated by a thin layer of fibrous tissue. With respect to the capsule composed of fibroblsts and fibrocytes, fibroblasts began to appear on the third day, increased to a maximum on the seventh day, and decreased at fourteen days. In contrast, fibrocytes continued to increase from three days to fourteen days. The encapsu-lation of implant material with connective tissue may be considered to be the ideal tissue-implant relation, or may be a good criterion for biocompatibility (2, 11, 12), that is, the results obtained so far suggest that Bioceram® seems to be a satisfactorily biocompatible material.
It is thought that FBGCs are formed through fusion of activated macrophages (13, 14). Macrophages usually appear with inflammatory reaction, but not all the macrophages participate in FBGCs formation. It is generally understood that FBGCs are formed in case of strong foreign body properties of the bioimplant. Result that only few FBGCs (0.2%) were observed in all the specimens (Table1) after implantation might show that foreign body properties of Bioceram is low.
Some investigators have reported that bioceramic materials such as hydroxyapatite did not induce bone fomation when implanted in soft tissue (3, 15, 16). In our study of tissue reaction to a bioimplant in the early stage, we also did not observe any osteogenesis. As for there will be osteogenesis or not in the long term implantation of Bioceram® in the subcutaneous tissues, it is currently under observation.

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Received for publication December 2, 1997 ; accepted January 5, 1998.

1 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:+81-886-33-7208.