Histochemical detection of acid and alkaline phosphatases in periodontal tissues after experimental tooth movement

Acid and alkaline phosphatases were demonstrated histochemically in periodontal structures and alveolar bone tissues after experimental tooth movement. Previous reporting has concerned periodontal structures during orthodontic tooth movement (C. SANDSTEDT, Nord Tandl Tidsk No. 4,5, 1905; A.E. ZAKI and G.V. HUYSEN, JDent Res 42:1373-1379, 1963). Sixty Wistar strain rats were used in this experiment. During ether anesthesia, a rubber dam (1.5 mm wide and 0.15 mm thick) was inserted between the first and second molars on the left side of the upper jaw. The right side was observed as a control (K. TAKIMOTO ET AL: | Dent Res 45:1473-1476, 1966). Rats were killed in groups of 20 after one, three, and five days. In rats killed after five days, the rubber dam was replaced after three days by two rubber strips to exert a constant amount of force. Blocks of fresh tissue were prepared and decalcified for one week at 4C in a 10% solution of (tetrasodium salt of ethylendiamintetraacetate (EDTA), as a neutral solution adjusted to pH 7.0 with 5M citric acid. After rinsing in disstilled water, blocks were sagittally sectioned at 18,u in a cryostat with a sliding microtome. Serial sections were fixed (10% neutral formalin, 30 min) and stained with hematoxylin and eosin. The substrate solution for acid phosphatase consisted of 5 mg of naphthol AS-TR phosphate, dissolved in 0.1 ml of N-N dimethylformamide with 30 ml of 0.1M acetate buffer at pH 5.8 and added to 30 mg of fast red violet LB.* In sections incubated 1 to 1.5 hours at 37C, dark purple precipitation indicated enzyme-positive sites. The substrate solution for alkaline phosphatase consisted of 5 mg of naphthol AS-MX phosphate, dissolved in 0.1 ml of NCN dimethylformamide and 30 ml of Clark and Lub's buffer (K. KAWAKATSU and M. MORI, Cancer Res 23:539545, 1963) at pH 9.2 and added to 30 mg of fast red violet LB. After 30 to 50 minutes at 20C, enzyme-positive sites appeared red on a yellowbrown background. In normal tissues, moderate amounts of acid and alkaline phosphatases were observed in the periodontal ligaments. Alveolar bone surface and