The digestion of phagocytosed collagen is inhibited by the proteinase inhibitors leupeptin and E-64

Using morphometric methods the effects of the thiol-proteinase inhibitors leupeptin and E-64 on the digestion of intracytoplasmic collagen fibrils were studied in cultured mouse bone explants. Both drugs caused a dose-dependent increase of lysosomal structures containing cross-banded collagen fibrils (CCV) in periosteal fibroblasts. After an incubation period of 48 hours, leupeptin (in a concentration of 65 microM) caused a thirty-fold increase in the volume fraction of CCV. This effect proved to be reversible following upon the withdrawal of the drug. Since the leupeptin-related accumulation of intracellular collagen fibrils was not significantly inhibited by alpha, alpha dipyridyl (a drug that interferes with collagen fibril formation), it is thought unlikely that the fibrils represented newly synthesized collagen. This view is further substantiated by data obtained from explants incubated in the presence of the phagocytosis-inhibiting agent cytochalasin B. This compound completely inhibited the leupeptin-related accumulation of CCV. The data strongly suggest that collagen fibrils found in cytoplasmic vacuoles of periosteal fibroblasts represent collagen taken up by phagocytosis, the integrity of cytoplasmic actin filament systems is a prerequisite for phagocytosis of collagen to occur, and thiol-proteinases, such as cathepsin B, L, and/or N, play an essential role in the digestion of internalized collage

Transmission electron microscopy of bone

This chapter describes procedures to process mineralized tissues obtained from different sources for transmission electron microscopy (TEM). Methods for fixation, resin embedding, staining of semi-thin sections and ultrathin sections are presented. In addition, attention will be paid to processing of cultured bone explants for TEM analysis

Endocytosis of tartrate-resistant acid phosphatase by osteoblast-like cells is followed by inactivation of the enzyme

Tartrate-resistant acid phosphatase (TRACP) is generally used as a marker of osteoclasts. Yet, other bone-associated cells, such as osteoblasts and osteocytes, may also express activity of this enzyme. Osteoblasts containing TRACP activity are seen particularly in the vicinity of areas of bone resorption, suggesting that osteoclasts somehow induce TRACP activity in osteoblasts. In a recent study, we found that osteoblast-like cells appeared to have the capacity to endocytose TRACP released by osteoclast precursors. In the present study, we investigated the endocytosis of TRACP in more detail as well as the fate of the endocytosed enzyme. We found that incubation of osteoblast-like cells with TRACP-coated beads resulted in attachment of a high number of beads to the cells. After culturing osteoblast-like cells with medium conditioned by blood monocytes that contain TRACP, activity of the enzyme was found in the cells. Following replacement of the medium by normal medium that did not contain TRACP, a decrease in the level of TRACP activity in osteoblast-like cells occurred. Our data strongly suggest that osteoblast-like cells recognize TRACP released by osteoclast precursors and that upon endocytosis inactivation of the enzyme occurs. We propose that uptake of the enzyme is important for the control of enzyme activity, thereby preventing degradation of matrix constituent

Transmission electron microscopy of bone

Electron microscopic analysis of mineralized tissues like bone and dentin is essential for understanding of cell–cell/cell–matrix interactions, and the three-dimensional organization of these tissues. This chapter describes a few methods to process mineralized tissues obtained from different sources for ultrastructural analysis by transmission electron microscopy

Transmission electron microscopy of bone

This chapter describes procedures to process mineralized tissues obtained from different sources for transmission electron microscopy (TEM). Methods for fixation, resin embedding, staining of semi-thin sections and ultrathin sections are presented. In addition, attention will be paid to processing of cultured bone explants for TEM analysis

Plasminogen activators are involved in the degradation of bone by osteoclasts

Osteoclastic bone degradation depends on the activity of several proteolytic enzymes, in particular to those belonging to the classes of cysteine proteinases and matrix metalloproteinases (MMPs). Yet, several findings suggest that the two types of plasminogen activators (PA), the tissue- and urokinase-type PA (tPA and uPA, respectively) are also involved in this process. To investigate the involvement of these enzymes in osteoclast-mediated bone matrix digestion, we analyzed bone explants of mice that were deficient for both tPA and uPA and compared them to wild type mice. The number of osteoclasts as well as their ultrastructural appearance was similar for both genotypes. Next, calvarial and metatarsal bone explants were cultured for 6 or 24 h in the presence of selective inhibitors of cysteine proteinases or MMPs and the effect on osteoclast-mediated bone matrix degradation was assessed. Inhibition of the activity of cysteine proteinases in explants of control mice resulted in massive areas of non-digested demineralized bone matrix adjacent to the ruffled border of osteoclasts, an effect already maximal after 6 h. However, at that time point these demineralized areas were not observed in bone explants from uPA/tPA deficient mice. After prolonged culturing (24 h), a comparable amount of demineralized bone matrix adjacent to actively resorbing osteoclasts was observed in the two genotypes, suggesting that degradation was delayed in uPA/tPA deficient bones. The activity of cysteine proteinases as assessed in bone extracts, proved to be higher in extracts from uPA/tPA−/− bones. Immunolocalization of the integrin αvβ3 of in vitro generated osteoclasts demonstrated a more diffuse labeling of osteoclasts derived from uPA/tPA−/− mice. Taken together, our data indicate that the PAs play a hitherto unrecognized role in osteoclast-mediated bone digestion. The present findings suggest that the PAs are involved in the initial steps of bone degradation, probably by a proper integrin-dependent attachment to bone