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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The most obvious proteolytic event controlled by the osteoclast is bone matrix removal in the resorption compartment. Here, however, we investigated whether matrix metalloproteinase (MMP) activity of the osteoclast might be involved in its migration to its future bone resorption site. We seeded either nonpurified or purified osteoclasts onto either uncoated or collagen-coated dentine slices and cultured them in the presence or absence of specific MMP inhibitors. When nonpurified osteoclasts were cultured on uncoated dentine, MMP inhibitors did not prevent pit formation, as previously reported. However, when collagen-coated dentine was used, pit formation was strongly inhibited by MMP inhibitors. The same results were obtained when performing these experiments with purified osteoclasts, thus demonstrating the ability of osteoclasts by themselves to migrate through collagen via an MMP-dependent pathway. This demonstration was confirmed by using collagen-coated invasion chambers. In addition, the invasions were not, or only slightly, inhibited by inhibitors of serine proteinases, cysteine proteinases, and carbonic anhydrase, though the latter two are well established bone resorption inhibitors that strongly inhibited pit formation. It is concluded that osteoclasts can migrate through collagen in the absence of other cells and that this migration relies on MMP activity, whereas other enzymes typically required for bone removal in the resorption compartment are not essential for migration. Some of the osteoclast MMPs might thus be relevant to the migratory/invasive activity of the osteoclast, rather than to its bone resorptive activity itself.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The access of the osteoclast to the bone surface is an early determinant of bone resorption and has important implications for bone morphogenesis, repair, and maintenance. Besides growth factors, cytokines, and chemoattractants, this process involves a series of cell–matrix and cell–cell interactions, leading to distinct osteoclast activities, such as invasion, migration, and anchorage to the bone surface.(1)

Ultrastuctural studies first suggested that the osteoclast was likely to interact with unmineralized matrix before establishing the bone resorption compartment on the bone surface.(2,3) The characterization of specific receptors on the plasma membrane of the osteoclast helped to identify the matrix constituents that might be critical for these interactions: e.g., type I collagen, the major protein of the bone matrix(4); RGD (Arg-Gly Asp) peptide-containing proteins, such as vitronectin, osteopontin, and bone sialoprotein(5); hyaluronate(6); or others like matrix-bound macrophage colony-stimulating factor (M-CSF)(7) and the C3 component of the complement.(8)

Though it has not been directly investigated whether these cell matrix interactions result in specific enzyme requirements for osteoclast migration, several lines of evidence indicate that matrix metalloproteinases (MMPs) may be critical for the access of osteoclasts to their future resorption sites. First, MMP activity proved indispensable for the migration of osteoclasts to the developing marrow cavity of primitive long bones.(9) This process concerned invasion through osteoid and involved only focal lysis as it proceeded concomitantly with osteoid maturation and the development of a bone collar. The expression of MMP-9 in the migrating osteoclasts and of MMP-13 in the hypertrophic chondrocytes, localized in the central cores of these bones, led the authors to speculate that MMP-9 of osteoclasts might play a direct role in this migration and/or that MMP-13 of chondrocytes might release collagen fragments that are chemotactic to the osteoclasts. Second, the recent identification of MT1-MMP in invadopodia and lamellipodia of osteoclasts suggested a role for this membrane-bound proteinase in osteoclast invasion/migration.(10) Earlier it was proposed that proteases control the access of the osteoclast to the bone surface.(11) It was hypothesized, however, that these proteinases were MMPs of osteoblasts clearing the osteoid from the bone surfaces.(12) This view was supported by the demonstration that osteoblasts release MMPs in response to bone-resorbing agents(13,14) as well as by recent in vivo observations.(15) Thus, there are several indications that MMPs are important for the recruitment of osteoclasts to future resorption sites, but their mode of action is unclear and it is not known what is the main cell conducting these proteolytic events.

There are many physiological situations where MMP activity of the invading/migrating cell itself is involved in the focal proteolysis required for its invasion/migration.(16-19) However, the possibility that the osteoclast might use MMPs for its invasive/migratory activity was never made clear. In contrast, much attention was paid to the proteolytic events directed by the osteoclast to remove bone matrix, once it had established the resorption zone. This led to the identification of a series of MMPs in the osteoclasts(10,20-25) as well as of cysteine proteinases, the primary of which is now considered cathepsin K.(26-29) While the participation of cysteine proteinases in bone removal was shown in a series of conditions,(30) that of MMPs appears to depend on the experimental/physiological situation. Antibodies recognizing interstitial collagenase (MMP-1/MMP-13) stained the resorption zone,(20) and electron microscopy showed unambiguously that MMP inhibitors inhibit collagen degradation in the subosteoclastic resorption zones of mouse calvariae.(31) However, MMP inhibitors appeared rather inefficient in inhibiting the resorptive activity of osteoclasts cultured on bone or dentine slices because the levels of inhibition ranged from 0%(32,33) to 30%.(23)

In the present study, we examined whether MMP activity exerted by the osteoclast mediates its access to the bone surface. We therefore developed assays allowing us to assess the invasion of purified osteoclasts into collagen and tested the effect of proteinase inhibitors on this process as compared with bone resorption. We found that osteoclasts invade collagen in the absence of other cells by using MMP activity and without requiring significantly the cysteine proteinase activity which is essential for bone removal in the resorption compartment.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Materials

Eight-day-old New Zealand rabbits were purchased from Statens Seruminstitut (Copenhagen, Denmark). Collagen solution (type I collagen extracted in acid from porcine tendons), bacterial collagenase, and pronase E were obtained from Nitta Gelatin Co. (Osaka, Japan), Wako Pure Chemical Co. (Osaka, Japan), and Kaken Chemical Co. (Tokyo, Japan), respectively. Cell culture inserts (12 mm) containing a polycarbonate membrane with 12 μm pores were from Costar (Cambridge, MA, U.S.A.). MMP inhibitors RP59794(34) and BB94(35) were kind gifts from Drs. Y. Lelievre and C.G. Caillard (Rhône-Poulenc Rorer, Vitry sur Seine, France) and Dr. H. Van Wart (Roche, Palo Alto, CA, U.S.A.), respectively. They are general MMP inhibitors with IC50's ranging between 10−9 and 10−8 M for MMP-1, MMP-3, and MMP-9(34,35) (unpublished data), each representing a distinct subgroup within the MMP family. Tissue inhibitor of metalloproteinase 2 (TIMP-2) was a kind gift of Dr. K. Langley (Amgen, Thousand Oaks CA, U.S.A.). Ivory dentine slices were a kind gift from Prof. M. Kumegawa (Meikai University, Saitama, Japan). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.).

Preparation of an unfractionated population of bone cells

Unfractionated populations of bone cells were prepared as reported.(36) Briefly, long bones of an 8-day-old rabbit were minced in alpha-modified essential medium (α-MEM) and agitated gently for 30 s with a vortex mixer. After sedimentation of bone fragments under normal gravity for 1 minute, the supernatant was harvested and washed twice by centrifugation at 100g for 2 minutes The cells were resuspended in α-MEM containing 5% fetal bovine serum (FBS).

Purification of osteoclasts

Osteoclasts were purified according to a published method(37) that we modified slightly. Eight milliliters of type I collagen solution (3 mg/ml) was mixed with 1 ml of 10× concentrated α-MEM and 1 ml of 0.2 M HEPES/NaOH (pH 7.4) containing 2.2% NaHCO3. Three milliliters of this mixture was poured into a 100-mm culture dish and incubated for 30 minutes at 37°C to allow polymerization of the gel. An unfractionated population of bone cells (1 × 108 cells) was then plated onto a collagen-coated dish and cultured in 5% CO2 incubator at 37°C. After 3 h of cultivation, nonadherent cells and small bone fragments were removed by washing extensively with phosphate-buffered saline (PBS) and treating with PBS containing 0.001% pronase E and 0.02% EDTA at 37°C for 15 minutes. Then, stromal cells were removed by a treatment for 10 minutes with 0.02% bacterial collagenase in PBS at room temperature, followed by washes with PBS. Finally, the osteoclasts were released from the gel by treatment for 15 minutes with 0.1% collagenase in PBS at 37°C. Released cells were pelleted by centrifugation at 100g for 2 minutes, resuspended in PBS, and pelleted by centrifugation at 400g for 3 minutes. The cells were treated for 5 minutes with PBS containing 1 mM EDTA, washed three times with α-MEM, and used as purified osteoclasts. To evaluate the osteoclast yield and purity, 50 μl of cell suspension was spotted in a 35-mm culture dish and incubated for 10 minutes, followed by the addition of 2 ml of α-MEM containing 5% FBS. The cells were then counted under a phase-contrast microscope and counted again after overnight culture to better evaluate their purity. For the latter evaluation, the cells were stained for tartrate-resistant acid phosphatase (TRAP) activity with a leukocyte acid phosphatase staining kit (Sigma). Our preparations provided cell populations where more than 97% of the cells were multinucleated and TRAP positive. When seeded on dentine slices, they generated pits as is typical for osteoclasts. Moreover, a characterization of these cells was performed previously and demonstrated all the typical features of the osteoclasts,(37) such as calcitonin receptors, a ruffled border and a sealing zone, and a high rate of excavation of dentine and bone in the absence of other cells, which could be further stimulated by cocultivation with stromal cells, and still further stimulated by cocultivation in the presence of 1,25-dihydroxyvitamin D3.

Pit assay

Dentine slices (diameter 6 mm, thickness 0.15 mm) were distributed in a 96-well plate. A collagen solution prepared as described above was diluted to 1.5 mg/ml with α-MEM and used for coating the dentine slices (10 μl/slice). The collagen was allowed to polymerize by incubating for 30 minutes at 37°C. An unfractionated population of bone cells was then seeded onto either uncoated or collagen-coated dentine slices (5 × 104 cells/slice) and cultured overnight in α-MEM containing 5% FBS and the indicated concentration of MMP inhibitor. Similar experiments were performed with purified osteoclasts (500 cells/slice). At the end of the culture, the cells and the collagen coat were scraped off, and the pits were stained with acid hematoxylin for 20 minutes. Pit areas were determined under a microscope by counting the number of mesh squares (10 × 10 μm) covering the pits.

Invasion assay in cell culture inserts

Invasion assays were performed according to published methods.(38) The upper surface of the membrane of each culture insert was coated with 30 μl of diluted collagen solution (1.5 mg/ml), and the collagen was allowed to polymerize at 37°C. The homogeneity of collagen coating was checked at this stage (and also after the culture) by staining with Coomassie Brilliant Blue R250. Purified osteoclasts (300–500 cells/insert) were spotted in the center of the insert in 50 μl of α-MEM. After 15 minutes, 1 ml of α-MEM containing the indicated enzyme inhibitor was added into the lower well (12-well plate) and 250 μl of the same medium was added into the insert. After overnight culture, the cells were stained for TRAP. In preliminary experiments, total osteoclast numbers were counted, and it was checked that the inhibitors did not affect the total number of osteoclasts. Next, the collagen and the osteoclasts located on the upper surface of the membrane were scraped away so that only the osteoclasts on the lower surface of the membrane or osteoclast extensions in the pores of the membrane were visible. Osteoclast invasion was evaluated under the microscope (20× objective) by counting the number of pores covered by osteoclasts or containing osteoclast extensions. Note that this evaluation does not discriminate whether extensions in pores next to each other are from the same or from distinct osteoclasts.

Statistical analysis

Data are expressed as the mean ± standard deviation (SD) of four cultures. Statistical differences between groups were evaluated by analysis of variance (ANOVA) with Scheffe's F-test. Differences were considered significant at p < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Nonpurified osteoclasts seeded on a calcified matrix produced resorption pits irrespective of the presence of MMP inhibitor (Fig. 1) in accordance with previous studies.(32,33) In physiological situations, however, osteoclasts are likely to interact with molecules of the nonmineralized matrix, such as collagen, before reaching the calcified bone surface. Therefore, we investigated whether seeding the osteoclasts on a collagen-coated calcified matrix would affect pit formation and its sensitivity to the MMP inhibitor. We found that the presence of a collagen layer resulted in a decrease in pit formation and enabled the MMP inhibitor to inhibit this pit formation dose dependently (Fig. 1). This is in line with the concept that nonmineralized collagen on the bone surface may act as a barrier for the access of the osteoclast to the bone matrix(11,39) and that MMPs play a role in the access of the osteoclasts to the calcified bone surfaces.(9,12)

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Figure FIG. 1. Pit formation by an unfractionated population of bone cells: the effect of MMP inhibitor depends on the presence of a collagen coat on the dentine slices. An unfractionated population of bone cells was cultured overnight on dentine slices that were coated or not with collagen, and in the presence of the indicated concentrations of the MMP inhibitor RP59794. The pit areas were evaluated as explained in Materials and Methods, and are shown as means ± SD of four cultures. *Significant effect of the inhibitor (p < 0.05).

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Although it is widely hypothesized that osteoblast MMPs may render bone surfaces accessible to the osteoclast by removing osteoid, it is unclear whether there are situations where the osteoclast may gain access to the bone surface by using its own MMP activity. We investigated this possibility by repeating the above experiment with a preparation of purified osteoclasts contaminated by less than 3% other cells. We found that these purified osteoclasts seeded on a calcified matrix and cultured overnight excavate pits, as was reported originally by Kakudo et al.(37) (Fig. 2). More importantly, they still do so when seeded on collagen-coated slices, and only inthis case do MMP inhibitors impede pit formation (Fig. 2). These observations show that the osteoclasts themselves can overcome a collagen barrir and gain access to the bone surface and that MMPs are rate-limiting for this process.

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Figure FIG. 2. Pit formation by purified osteoclasts: the effect of MMP inhibitor depends on the presence of a collagen coat on the dentine slices. Purified osteoclasts were cultured overnight on dentine slices that were coated or not with collagen, and with or without 10 μM RP59794. Further experimental procedures and presentation of the data are as in Fig. 1.

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To investigate how the osteoclasts overcome the collagen barrier and to establish the exact role of MMPs in this process, we used cell culture inserts and examined the effect of MMP inhibitors on the invasion of collagen by purified osteoclasts. Preliminary experiments were performed on uncoated membranes and showed that after an overnight culture, a number of osteoclasts had extended cell processes through the pores of the membranes and were spread to various extents over the lower surface of the membranes. The MMP inhibitor did not affect this process, thus showing that this drug does not interfere with cell locomotion itself (Fig. 3A). Next, the purified osteoclasts were seeded on collagen-coated membranes and cultured overnight. The collagen coat was not affected by this culture, as evaluated by Coomassie Blue staining. Despite this collagen barrier, many cells extended processes in the pores of the membrane or were spread to some extent over the lower surface, thus indicating that the osteoclasts were able to project cell processes and move through the collagen coat (Fig. 3B). Importantly, the MMP inhibitor inhibited the latter process dose dependently (Fig. 3A). Similar inhibitions were obtained with BB-94, another synthetic MMP inhibitor that has been used to assess the role of MMPs in a number of investigations(35) (Fig. 4). Also TIMP-2, a physiological MMP inhibitor, used at 10 μg/ml, brought the invasions down to 51 ± 6% of the control value (four control and four TIMP-2 cultures). These experiments indicate that the osteoclasts overcome the collagen barrier by migrating through it, via an MMP-dependent pathway which is not involved, however, in osteoclast locomotion itself.

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Figure FIG. 3. Invasion of collagen by purified osteoclasts. (A) Dose-dependent effect of MMP inhibitor. Purified osteoclasts (300 cells/insert) were cultured overnight onto filters that had been coated or not with collagen, and in the presence of the indicated concentrations of RP59794. The invasions were evaluated as explained in Materials and Methods and are shown as means ± SD of four cultures. *Significant effect of the inhibitor (p < 0.05). (B) Appearance of the lower surface of a membrane after a culture of purified osteoclasts on the collagen-coated upper surface. Pictures were taken through the microscope after an overnight culture in the (a, b) absence or in the (c) presence of 10 μM RP59794, and after removal of the cells and the collagen layer from the upper surface. Arrowheads indicate osteoclast invasions. Bars in (a), 50 μm; in (b) and (c), 300 μm.

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Figure FIG. 4. Invasion of collagen by purified osteoclasts: effect of inhibitors of different classes of proteinases and of carbonic anhydrase. Purified osteoclasts (500 cells/insert) were cultured overnight on collagen-coated filters in the presence or absence of inhibitors, as indicated. Further experimental procedures and presentation of the data are as in Fig. 3A.

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Finally, we evaluated the relative importance of the role of MMPs in this process, by comparing the effect of MMP inhibitors to that of inhibitors of other enzymes. Plasminogen activator was identified in osteoclasts(40-42) and was implicated in various situations of cell invasion.(43) However, aprotinin, an inhibitor of the plasminogen activator/plasminogen cascade, was without influence on collagen invasion by the osteoclast (Fig. 4) Cathepsin K, a cysteine proteinase, is highly expressed in osteoclasts(26,28) and cysteine proteinases are necessary for the collagenolytic activity in the resorption zone of the osteoclast.(31) When we tested E-64, an inhibitor of cysteine proteinases on osteoclast invasion in collagen, we found only a slight inhibitory effect as compared with the inhibitions obtained with MMP inhibitors, suggesting that the participation of cysteine proteinases in this process was minor as compared with that of MMPs (Fig. 4) The low inhibitory activity of E-64 could not be ascribed to its limited plasma membrane permeability, because EST (also called Ep-453), an analog of E-64 that penetrates much faster in the cells,(44) was even less inhibitory when tested at the same concentration as E-64 (data not shown). Carbonic anhydrase is a key enzyme of the resorptive activity of osteoclasts, and accordingly, ethoxyzolamide, its inhibitor, is a potent inhibitor of bone resorption.(45) Ethoxyzolamide was, however, without effect on invasion in collagen (Fig. 4) In contrast, when E-64 and ethoxyzolamide were tested at the same concentrations on pit formation (uncoated slices), we observed inhibitions of 50 ± 6% and 90 ± 7%, respectively (mean ± SD of four cultures for each experimental condition). Thus, in the present experimental system, MMPs appear to be the key proteinases for osteoclast invasion through collagen, whereas bone resorption itself appears to rely instead on cysteine proteinases.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The recruitment of osteoclasts to future resorption sites is controlled by a mechanism where cell–matrix interactions play an important role. Type I collagen is likely to be one of the organic matrix molecules involved in these interactions because the osteoclast has specific receptors for collagen,(4) collagen is very abundant in bone, and indications exist that collagen affects the access of the osteoclast to the bone surface(11,39) (and present data). While it is often believed that collagen removal by osteoblasts or bone-lining cells is a prerequisite for bone resorption, we show here that the osteoclast itself can migrate through type I collagen and requires MMP activity for this migration.

The evidence for MMP requirement for migration through collagen is based on the inhibition of this process by MMP inhibitors. It is likely that the primary target of these inhibitors was really MMP activity, since similar results were obtained with two distinct synthetic inhibitors of MMP activity, as well as with TIMP-2, an endogenous inhibitor of MMP activity whose selectivity for MMPs is still higher than that of the synthetic inhibitors.(46) Furthermore, these drugs did not show toxic effects and appeared to inhibit fairly specifically the migration through collagen, since they did not affect other osteoclast activities. Locomotion itself was unaffected because the osteoclasts migrated to the lower surfaces of the uncoated membranes irrespective of the presence of MMP inhibitors. The resorptive activity was not affected either because pit formation in uncoated dentine was insensitive to the inhibitors, which is in line with previous observations.(32,33) It is thus unlikely that the inhibitions of migration are merely a consequence of a general disorder caused by the MMP inhibitors. It is also unlikely that the ability of purified osteoclasts to migrate through collagen results from a pecularity related to the purification procedure because unpurified osteoclasts showed the same ability to migrate through collagen via an MMP-dependent pathway.

That the MMP activity of the osteoclast itself is able to prompt the migration of the osteoclasts through collagen is compatible with the fact that osteoclasts express various MMPs. These include MMP-9,(9,21-25,47) MT1-MMP,(10) and MMP-12.(47,48) MMP-1/MMP-13,(20,23-25,49) MMP-3,(20,23) and MMP-2(23) were also reported in osteoclasts, but there were also situations where it was not possible to detect MMP-1/MMP-13,(9,33,50) MMP-3,(24,25) and MMP-2.(24) It could be that some of the latter proteinases are synthesized by nonosteoclastic cells, get adsorbed subsequently onto the surface of the osteoclast, and remain there even after purification of the osteoclasts. The latter may apply to MMP-2, which is highly expressed by cells of the osteoblast lineage(51) and can bind to αvβ3 integrins,(52) which are abundant on the osteoclast membrane.(53) Therefore, we cannot exclude an indirect participation of nonosteoclastic cells to the migration of the osteoclasts through collagen. Our work shows, however, that osteoclast-directed MMP proteolysis (whether these MMPs are of osteoclastic origin or not) is sufficient for their migration through collagen, and that the access of the osteoclast to the surface of the mineralized matrix does not necessarily require removal of collagen from the bone surfaces by osteoblasts or lining cells.(12) Note that collagen clearance was also not seen in the physiological situation where (pre)osteoclasts migrate through osteoid on their way to the developing marrow cavity of primitive long bones.(9)

It is of interest that the MMPs expressed in osteoclasts were also implicated in the invasive/migratory activities of other cell types. MMP-9 has been implicated in the invasion of trophoblasts into the placental wall,(16) in keratinocyte migration during wound healing,(17) in invasion of lymphocytes into the basement membrane,(18) and in tumor invasion.(19) MMP-12 is necessary for the invasion of macrophages into basement membranes.(54) MT1-MMP is expressed in invasive cells, such as endothelial cells(55) and trophoblasts.(56) Furthermore, in osteoclasts, this MT1-MMP has been localized(10) on cell extensions specialized in adhesions of short duration,(1) typically involved in invasive events,(57) so that osteoclasts might well use MT1-MMP to perform focal proteolysis and move through the extracellular matrix.(10)

It should be stressed that the role of MMPs in the migration of osteoclasts through collagen appears unique among various proteinases because there was no evidence for an involvement of serine proteinases and only a weak participation of cysteine proteinases. It is interesting that cell culture experiments show that in contrast, pit formation in the mineralized bone matrix itself appears to rely much more on cysteine proteinases than on MMPs(23,32,33,58-60) (and present data), even when bone resorption is assessed through collagen degradation in the resorption pits.(33) These distinct proteolytic requirements for migratory and resorptive functions may be related to the fact that migrating and resorbing osteoclasts differ in cell shape, organization of the cytoskeleton, the attachment apparatus, and the functional domains of the plasma membrane,(1,61) as well as in expression of enzymes such as carbonic anhydrase.(62) Moreover, migration through collagen in the complete absence of mineral results in quite round osteoclasts(4) that do not show any polarization of the proton pump.(63) This is in marked contrast with the resorbing osteoclasts typically polarized as are epithelial cells,(1) even in the special situation where resorption concerns mainly collagen.(64) The present data showing that migration and resorption appear mediated by distinct proteolytic activities are thus in line with the view that distinct osteoclast activities require distinct functional and structural organizations. More particularly, they draw attention to the possibility that some osteoclast MMPs do not exert their activity in the resorption compartment, which is in line with the pH optimum of MMPs well above the acidic levels of the resorption compartment.

So far the physiological implications of the osteoclast–matrix interactions have been mainly related to cell attachment or to transmission of specific signals of the matrix to the cell.(1,4,5) The possibility that these osteoclast–matrix interactions result in specific enzyme requirements for osteoclast detachment and migration had not been investigated. The present data show that in situations where collagen is present on the bone surface, the osteoclast uses MMP activity to migrate and gain access to its future resorption site.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The authors are grateful to Dorte Larsen, for expert technical assistance, and to the authors of relevant papers that were not cited because of lack of space.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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