W. Beertsen

The Bone Lining Cell: Its Role in Cleaning Howship's Lacunae and Initiating Bone Formation

In this study we investigated the role of bone lining cells in the coordination of bone resorption and formation. Ultrastructural analysis of mouse long bones and calvariae revealed that bone lining cells enwrap and subsequently digest collagen fibrils protruding from Howships lacunae that are left by osteoclasts. By using selective proteinase inhibitors we show that this digestion depends on matrix metalloproteinases and, to some extent, on serine proteinases. Autoradiography revealed that after the bone lining cells have finished cleaning, they deposit a thin layer of a collagenous matrix along the Howships lacuna, in close association with an osteopontin‐rich cement line. Collagenous matrix deposition was detected only in completely cleaned pits. In bone from pycnodysostotic patients and cathepsin K‐deficient mice, conditions in which osteoclastic bone matrix digestion is greatly inhibited, bone matrix leftovers proved to be degraded by bone lining cells, thus indicating that the bone lining cell “rescues” bone remodeling in these anomalies. We conclude that removal of bone collagen left by osteoclasts in Howships lacunae is an obligatory step in the link between bone resorption and formation, and that bone lining cells and matrix metalloproteinases are essential in this process.

Phagocytosis of bone collagen by osteoclasts in two cases of pycnodysostosis

SummaryElectron microscopic examination of bone biopsies obtained from two patients suffering from pycnodysostosis revealed that osteoclasts contained (sometimes large) cytoplasmic vacuoles filled with bone collagen fibrils. These vacuoles stained positive for acid phosphatase activity, thereby suggesting that bone matrix had been phagocytosed and subsequently exposed to hydrolytic enzymes of the lysosomal apparatus. Collagen-containing vacuoles were not observed in osteoclasts of individuals not suffering from this disease.

Alkaline phosphatase induces the mineralization of sheets of collagen implanted subcutaneously in the rat.

To determine whether alkaline phosphatase (ALP) can cause the mineralization of collagenous matrices in vivo, bovine intestinal ALP was covalently bound to slices of guanidine-extracted demineralized bovine dentin (DDS). The preparations were implanted subcutaneously over the right half of the rat skull. Control slices not treated with the enzyme were implanted over the left half of the skull of the same animals. Specimens were harvested after periods varying from 1 to 4 wk. It was shown that ALP-coupled DDS rapidly accumulated hydroxyapatite crystals. 4 wk after implantation, the content of calcium and phosphate per microgram of hydroxyproline amounted up to 80 and 60%, respectively, of that found in normal bovine dentin. Our observations present direct evidence that ALP may play a crucial role in the induction of hydroxyapatite deposition in collagenous matrices in vivo.

Cementum and Dentin in Hypophosphatasia

Hypophosphatasia (HPP) often leads to premature loss of deciduous teeth, due to disturbed cementum formation. We addressed the question to what extent cementum and dentin are similarly affected. To this end, we compared teeth from children with HPP with those from matched controls and analyzed them microscopically and chemically. It was observed that both acellular and cellular cementum formation was affected. For dentin, however, no differences in mineral content were recorded. To explain the dissimilar effects on cementum and dentin in HPP, we assessed pyrophosphate (an inhibitor of mineralization) and the expression/activity of enzymes related to pyrophosphate metabolism in both the periodontal ligament and the pulp of normal teeth. Expression of nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) in pulp proved to be significantly lower than in the periodontal ligament. Also, the activity of NPP1 was less in pulp, as was the concentration of pyrophosphate. Our findings suggest that mineralization of dentin is less likely to be under the influence of the inhibitory action of pyrophosphate than mineralization of cementum.

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 collagen.

Cathepsin K deficiency in pycnodysostosis results in accumulation of non-digested phagocytosed collagen in fibroblasts

The rare osteosclerotic disease, pycnodysostosis, is characterized by decreased osteoclastic bone collagen degradation due to the absence of active cathepsin K. Although this enzyme is primarily expressed by osteoclasts, there is increasing evidence that it may also be present in other cells, including fibroblasts. Since fibroblasts are known to degrade collagen intracellularly following phagocytosis, we analyzed various soft connective tissues (periosteum, perichondrium, tendon, and synovial membrane) from a 13-week-old human fetus with pycnodysostosis for changes in this collagen digestion pathway. In addition, the same tissues from cathepsin K-deficient and control mice were analyzed. Microscopic examination of the human fetal tissues showed that cross-banded collagen fibrils had accumulated in lysosomal vacuoles of fibroblasts. Morphometric analysis of periosteal fibroblasts revealed that the volume density of collagen-containing vacuoles was 18 times higher than in fibroblasts of control patients. A similar accumulation was seen in periosteal fibroblasts of three children with pycnodysostosis. In contrast to the findings in humans, an accumulation of internalized collagen was not apparent in fibroblasts of mice with cathepsin K deficiency. Our observations indicate that the intracellular digestion of phagocytosed collagen by fibroblasts is inhibited in humans with pycnodysostosis, but probably not in the mouse model mimicking this disease. The data strongly suggest that cathepsin K is a crucial protease for this process in human fibroblasts. Murine fibroblasts may have other proteolytic activities that are expressed constitutively or up regulated in response to a deficiency of cathepsin K. This may explain why cathepsin K-deficient mice lack the dysostotic features that are prominent in patients with pycnodysostosis.

Alkaline Phosphatase Induces the Deposition of Calcified Layers in Relation to Dentin: An in vitro Study to Mimic the Formation of Afibrillar Acellular Cementum

An attempt was made to test the hypothesis that alkaline phosphatase, an enzyme which is abundant in periodontal ligament, plays a role in the formation of acellular root cementum. Thin slices of bovine dentin were incubated in Iscove Modified Dulbeccos Medium supplemented with 10% normal rabbit serum and 10 mmol/L (3-glycerophosphate (P-GP) or folded into pericardial explants. Intestinal bovine alkaline phosphatase (APase), covalently linked to agarose beads, was added to the cultures. In the presence of the enzyme, the dentin slices were covered with thin layers of mineralized material. Such layers were not observed in cultures not provided with APase-beads or β-GP. They also did not form in relation to demineralized dentin. The layers of calcified material appeared to consist of crystallites embedded in a granular matrix of moderate electron density, which often exhibited the presence of incremental lines and resembled the matrix of afibrillar acellular cementum formed under in vivo conditions. When pericardial explants were interposed between the enzyme-containing beads and the dentin, mineral deposition in relation to the dentin was retarded. This finding lends support to the view that soft connective tissues interfere with the free diffusion of phosphate.

(Pre-)Osteoclasts Induce Retraction of Osteoblasts Before Their Fusion to Osteoclasts†

Precursors of osteoclasts seeded on top of a confluent layer of osteoblasts/bone lining cells induced retraction of the latter cells. The (pre)osteoclasts then migrated in the formed cell‐free areas and fused to form osteoclast‐like cells. Retraction of the osteoblasts/bone lining cells proved to depend on activity of matrix metalloproteinases, and TGF‐β1 prevented the retraction.

A Quantitative Enzyme Histochemical Analysis of the Distribution of Alkaline Phosphatase Activity in the Periodontal Ligament of the Rat Incisor

The spatial distribution of alkaline phosphatase (ALP) activity was examined in the periodontal ligament of the continuously growing rat incisor. With the indoxyl-tetrazolium salt method, enzyme activity was demonstrated in undecalcified cryosections, and the amount of reaction product was quantified. ALP activity appeared to be distributed heterogeneously. Its highest activity was found in the bone-related compartment of the ligament. In the tooth-related compartment and the supracrestal extension of the ligament, enzyme activity was significantly lower, but still higher than in the lamina propria of the gingiva. In the part of the ligament bordering the cementum, highest activity was found in the apical region just occlusal to Hertwigs epithelial root sheath, where formation of acellular cementum begins. From there toward the incisal edge, the activity of the enzyme gradually decreased. It is suggested that differences among the various parts of the periodontal ligament are related to local variations in phosphate metabolism and cementum deposition.Received for publication December 11, 1992 Accepted for publication March 17, 1993

Calvarial Osteoclasts Express a Higher Level of Tartrate-Resistant Acid Phosphatase than Long Bone Osteoclasts and Activation Does not Depend on Cathepsin K or L Activity

Bone resorption by osteoclasts depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Next to these enzymes, tartrate-resistant acid phosphatase (TRAP) is considered to participate in this process. TRAP is synthesized as an inactive proenzyme, and in vitro studies have shown its activation by cysteine proteinases. In the present study, the possible involvement of the latter enzyme class in the in vivo modulation of TRAP was investigated using mice deficient for cathepsin K and/or L and in bones that express a high (long bone) or low (calvaria) level of cysteine proteinase activity. The results demonstrated, in mice lacking cathepsin K but not in those deficient for cathepsin L, significantly higher levels of TRAP activity in long bone. This higher activity was due to a higher number of osteoclasts. Next, we found considerable differences in TRAP activity between calvarial and long bones. Calvarial bones contained a 25-fold higher level of activity than long bones. This difference was seen in all mice, irrespective of genotype. Osteoclasts isolated from the two types of bone revealed that calvarial osteoclasts expressed higher enzyme activity as well as a higher level of mRNA for the enzyme. Analysis of TRAP-deficient mice revealed higher levels of nondigested bone matrix components in and around calvarial osteoclasts than in long bone osteoclasts. Finally, inhibition of cysteine proteinase activity by specific inhibitors resulted in increased TRAP activity. Our data suggest that neither cathepsin K nor L is essential in activating TRAP. The findings also point to functional differences between osteoclasts from different bone sites in terms of participation of TRAP in degradation of bone matrix. We propose that the higher level of TRAP activity in calvarial osteoclasts compared to that in long bone cells may partially compensate for the lower cysteine proteinase activity found in calvarial osteoclasts and TRAP may contribute to the degradation of noncollagenous proteins during the digestion of this type of bone.