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Summary: To detect S-phase cells in the decalcified paraffin-embedded temporomandibular joint (TMJ) tissues of the rat, bromodeoxyuridine (BrdUrd) immunohistochemistry was performed. Percardiac perfusion of 10% EDTA-2Na Tris buffer accelerated the decalcification. The use of the monoclonal antibody to BrdUrd enabled the visualization of the distribution of S-phase cells. This method is practical in studying cell kinetics and the specific proliferation mode of the TMJ.Immunohistochemical investigations using bromodeoxyuridine (BrdUrd) and its monoclonal antibody have played an important role in analyzing cell kinetics or differential cell growth (Sasaki et al., 1986;Tatematsu et al. , 1987). However, previous studies have dealt mostly with normal or pathological conditions of the soft tissues such as tumours.Approaches to hard tissues have been previously performed to detect the chondrocyte proliferation in cartilage (Apte, 1988), undecalcified, plasticembedded bone (Apte et al. , 1990) , and decalcified jaw or tooth (Hume et al., 1990). A study by Gilhuus-Moe (1971) with tritiated thymidine (3H-TdR) autoradiography provided some knowledge on the growth of the TMJ. However, 3H-TdR autoradiography method is complicated and its processing time is long. Furthermore, no report on the application of BrdUrd immunohistochemistry to the TMJ in order to study its cell kinetics has been found. To perform BrdUrd immunohistochemistry in paraffin-embedded hard tissue, decalcification is required prior to thin sectioning. As to the decalcification of hard tissues in immunohistochemical study, the difference of decalcifying fluids has been reported to affect the tissue processing time or the morphological quality in staining (Apte et al., 1989).This paper describes the immunohistochemical detection of S-phase cells in the decalcified paraffinembedded TMJ of the rat. Materials and MethodsMale Sprague-Dawley rats aged 6 weeks (n = 6, mean body weight: 175 g) were used. They were injected intraperitoneally with BrdUrd (Sigma Chemical Co. , MO) in the dose of 40 mg/kg as a solution of 10 mg/mi in phosphate buffered saline (PBS). One hour later, the thoracic cavity was exposed and percardiac perfusion with 0.5% heparinized saline (500 ml) was carried out immediately after sacrifice with intraperitoneal overdose of sodium pentobarbital. Two kinds of fixatives (70% ethanol, 1 L, 4°C and 10% neutral phosphate buffered formalin, pH: 7.3, 1 L, 4°C) were sequentially perfused.After fixation was completed in 2 rats, approximately 3 mm thick sections were cut in the frontal plane from the nasal apex to the TMJ region by a diamond band saw (Exakt, Germany). Thick sections were decalcified for 7 days in the solution of Plank Rychlo (1952) or 10% solution of EDTA-2Na in 0.1 M Tris buffer brought to pH 7.3 with KOH pellets at 4°C. In the other 3 rats, the decalcifying fluid (10% EDTA-2Na solution for two and Plank Rychlo's solution for one rat) was sequentially perfused after fixation. It was continued for 4 days (4 L/day, dripping rate; approx...

The aim of this study was to assess the healing response to the bone implants treated by heating or freezing. Allogenic calvarial implants treated by heating at 65˚C for 1 hour, 3 freezes of 3 minutes, or boiling for 10 minutes (65˚C, frozen and boiled group respectively) were tested for the potential to fill cranial bone defects of rabbits. Both the 65˚C and frozen groups similarly showed more than 60% of new bone formation in terms of the untreated control. Among the three treatment modalities, boiling presented the most remarkable impairment on the reparative capacity of the implants. The autogenous bone segments reimplanted to their original mandibular sites after treatment by heating at 65˚C using microwave or by freezing were revealed to similarly incorporate to the host bone in dog experiments. In summary, heat treatment at 65˚C for 1 hour is comparable with freeze treatment in terms of preservation of the regenerative potential of bone for simultaneous reimplantation.

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