An immuno-electron microscopic study of the localization of cathepsins B and L and collagenase in cultured human odontoclasts

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We isolated human odontoclasts from primary teeth and cultured them on the culture dishes or dentine slices with alpha-MEM and 10% FCS.Human odontoclasts were tertrate-resistant acid phosphatase positive and formed resorption lacunae on the dentine slices. We examined the effect ot 5 mU/ml calcitonin on the morphological changes of odontoclasts, but no morphological changes were observed. Because of the difficulty of the isolation of odontoclasts and lack of the supply of the samples (primary teeth), we could not obtain many data from cultured human odontoclasts. Further investigations are needed for finding the efficient-isolation techniques and an optimal condition for human odontoclasts.We also examined the changes in the immunocytochemical localization of cathepsin L,type I collagen and rat salivary cystatin-3 (RSC-3) in rat osteoclasts treated with E-64. The immunoreactivity for cathepsin L was extracellularly very weak, but was strong intracellurlarly in the E-64-treated osteoclasts. The intracellular immuno-reactivity for type I collagen was found inthe vacuoles in the E-64-treated osteoclasts but not in any vacuoles in the control osteoclasts. In the immunoreaction for RSC-3 in the E-64-treated, the cytoplasm of the ruffled border was positive, and also the tips of the RSC-3 positive-ruffled border appeared to deeply enter into the bone matrix. Intracellularly, the granular reaction products of RSC-3 were found in vacuoles. These finding suggests that in the E-64-treated osteoclasts, the extracellular release of cathepsin L was insufficient to suppress the degradation of collagen, and that RSC-3 is probably associated wit the inhibition of cathepsin L in the lysosomes in the osteoclasts.