Localisation of alkaline phosphatase in equine growth cartilage

The aim of this study was to localise alkaline phosphatase (ALP) activity in equine growth cartilage both histochemically and ultrastructurally. For histochemical studies, full thickness growth cartilage samples were obtained from 6 anatomical sites from 16 horses and ponies ranging in age from 90 d postconception to 12 years of age. For ultrastructural studies, samples were obtained from the lateral trochlear ridge of the distal femur of 3 animals ranging in age from 157 d postconception to 12 months of age. Alkaline phosphatase in histological sections was localised using a substituted naphthol reaction which revealed enzyme activity around the cell surfaces of hypertrophic chondrocytes. ALP activity was quantified by determining the labelling index of ALP positive chondrocytes. The labelling index depended on (1) the age of the animal (activity being highest in animals < 6 months old), and (2) the anatomical site (activity being lowest in the proximal phalanges and highest in the growth plate, reflecting the degree of long bone growth at each site). In order to demonstrate ALP activity ultrastructurally, two capturing agents were used: cerium and lead. This enzyme was visualised by the precipitation of electron-dense salts at the site of activity. ALP activity was localised at low levels on the surface membranes of chondrocytes in the midproliferative zone. The enzyme activity increased throughout the proliferative zone and was most intense on chondrocytes of the hypertrophic zone. ALP activity was seen to be concentrated on the matrix vesicles, the putative sites of mineralisation. This work demonstrates, for the first time in the horse, that ALP is a useful marker of incipient bone formation.