Calcif Tissue Int (1994) 55:368--375 Calcified Tissue International 9 1994 Springer-Vedag New York Inc. The Effects of Bisphosphonates on the Resorption Cycle of Isolated Osteoclasts K. Selander, P. Lehenkari, H. K. V/i~in/inen Department of Anatomy, University of Oulu, Kajaanintie 52 A, 90220 Oulu, Finland Received: 2 March 1994 / Accepted: 25 May 1994 Abstract: Binding sites for wheat germ agglutinin (WGA)- lectin have been shown to become revealed in the demin- eralized resorption lacunae that osteoclasts excavate on bone substrate. Peroxidase-conjugated WGA-lectin, which binds to bone matrix glycoconjugates and proteoglycans, was used in pit formation assays to assess the activity of isolated osteoclasts cultured on either 3-amino-1,1-hydroxy- propylidene-bisphosphonate (APD)-or dichloromethylene bisphosphonate (C12MBP)-covered bone slices. Immunoflu- orescence and histochemical techniques were also used to study the effects of bone-bound bisphosphonates on isolated rat osteoclasts. Neither APD nor ClzMBP interfered with the special organization of actin or vinculin in osteoclasts when the cells were initializing their resorption cycle. After 24 hours of culture, the number of resorbing osteoclasts in- creased strongly on control slices, but remained low on ei- ther APD- or Cl2MBP-treated slices. At this time, the actin and vinculin rings in osteoclasts also started to exhibit ab- normal, more diffuse staining. Both bisphosphonates studied resulted in signs of cytotoxicity: the number of osteoclasts decreased on APD- or ClaMBP-covered bone during the course of the study and those remaining attached exhibited severe cytoplasmic retractions. The total areas of resorption remained at significantly lower levels in both experimental groups studied, and this was due to decreases in both the number and sizes of individual resorption pits. The size of the most extensive lacunae detected on the C12MBP slices did not exceed 5 x 10 3 ~m 2, whereas on the control slices, resorption pits bigger than 15 x 10 3 p~m 2 were frequently discovered. Key words: Bisphosphonates -- Osteoclast -- Bone resorp- tion -- Actin -- Vinculin. Bone resorption by osteoclasts is a multistep process that is preceded by recruitment and fusion of mononuclear precur- sors into multinucleated cells. Mature osteoclasts attach to the bone surface and initiate demineralization by producing a subcellular, acidic milieu in the generation of which intra- cellular acid-producing machinery is essential (for review see ref [1]). Bisphosphonates, such as dichloromethylene bisphosphonate (CI2MBP) and 3-amino-l,l-hydroxypro- pylidene-bisphosphonate (APD) are potent antiresorptive agents that avidly bind to bone hydroxyapatite [2-4]. Their Correspondence to: K. Selander resorption inhibitive action is considered to occur via bind- ing to bone mineral and thereby inhibiting the action of os- teoclasts that engulf them [5, 6]. Potency discrepancy pre- vails between these two compounds in vivo and in vitro; APD is known to exceed ClzMBP in potency by an order of magnitude in vivo [7, 8]. Using in vitro assays, C12MBP has been shown to be a more effective inhibitor of bone resorp- tion and a potential cytotoxin in the presence of bone, whereas APD has been suggested to exert its action by in- terfering with events preceding resorption [9-13]. Resorption of bisphosphonate-covered bone has also been shown to lead to formation of irregularly shaped and shallow resorption pits, which may be difficult to detect [9, 13]. The action of mature osteoclasts in vitro can readily be investigated by the weU-established pit formation assay, in which the dissoluted area correlates with the activity of os- teoclasts [14, 15]. Analysis of the area resorbed in previous studies has been based on detection of the excavations by various stain- ing methods or on quantification of the number or areas of unstained pits by phase contrast or scanning electron mi- croscopy [16-22]. Previous data from our laboratory suggest that resorption and migration phases alternate in the life of the osteoclast and that reorganization of microfilaments is obligatory for the cell to enter either one of these phases [23, 24]. In the present study, the effects of bone-bound bisphosphonates on isolated osteoclasts were studied using the pit formation as- say. The morphology of the osteoclasts was evaluated by staining the cells with tartrate-resistant acid phosphatase (TRAP), an enzyme generally accepted as a marker for os- teoclasts [25]. The special organization of actin and vinculin at different stages of the resorption cycle of osteoclasts was visualized using immunofluorescence techniques. In the present study, we also introduce a simple method for visualization of resorption pits by taking advantage of the binding of WGA-lectins to bone matrix glycoconjugates and proteoglycans, which become revealed in the demineralized lacunae [26, 27]. The total resorbed area can then be mea- sured by combining the light microscope image with a color- based automated image analysis system. Materials and Methods Bisphosphonates C12MBP(disodium clodronate tetrahydrate) and APD (3-amino-I, 1- hydroxypropylidenebisphosphonic acid, tetrasodium salt) were do- nated by Leiras Biomedical Research Center, Turku, Finland.