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Abstract

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

In human cancellous bone, osteoclastic perforations resulting from normal remodeling were generally considered irreversible. In human vertebral samples, examined by backscatter electron microscopy, there was clear evidence of bridging of perforation defects by new bone formation. Hence trabecular perforations may not be irreversible.

Introduction: Preservation of the trabecular bone microarchitecture is essential to maintain its load-bearing capacity and prevent fractures. However, during bone remodeling, the osteoclasts may perforate the platelike trabeculae and disconnect the structure. Large perforations (>100 μm) are generally considered irreversible because there is no surface on which new bone can be laid down. In this work, we investigated the outcome of these perforations on human vertebral cancellous bone.

Materials and Methods: Using backscatter electron microscopy, we analyzed 264 vertebral bone samples from the thoracic and lumbar spine of nine subjects (44–88 years old). Nine fields (2 × 1.5 mm) were observed on each block. Several bone structural units (BSUs) were visible on a single trabecula, illustrating a dynamic, historical aspect of bone remodeling. A bridge was defined as a single and recent BSU connecting two segments of trabeculae previously separated by osteoclastic resorption. They were counted and measured (length and breadth, μm).

Results and Conclusion: We observed 396 bridges over 2376 images. By comparison, we found only 15 microcalluses on the same material. The median length of the bridge was 165 μm (range, 29–869 μm); 86% being longer than 100 μm and 35% longer than 200 μm. Their breadth was 56 μm (range, 6–255 μm), but the thinnest were still in construction. Bridges were found in all nine subjects included in the study, suggesting that it is a common feature of normal vertebral bone remodeling. These observations support the hypothesis that perforation could be repaired by new bone formation. and hence, might not be systematically irreversible.


INTRODUCTION

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

Bone loss in elderly patients is presently understood as a imbalance between bone resorption and new bone formation, with progressive net loss over decades. In cancellous bone, this change in bone amount is associated with important structural changes. The key change in trabecular architecture seems to be rather a loss of entire structural elements (drop of Tb.N) than a progressive thinning of all the trabeculae (drop of Tb.Th). This has been observed initially on iliac crest biopsy specimens(1,2) and confirmed on vertebral trabecular bone.(3,4) The remodeling cycle (normal or pathological) is held responsible for that. During bone resorption, osteoclasts create cavities that may perforate the trabecula.(5) Such disruption of the trabecular network is also generally considered irreversible simply because new bone apposition can only occur on a pre-existing bone surface, and if the surface is removed (in case of perforation), the lost bone cannot be replaced by the normal remodeling process.(6) Consequently, perforations (i.e., larger than 100 μm) are generally considered as irreversible.

The dynamic of bone remodeling can be observed using various methods.(7,8) The backscatter electron microscopy (BSE) has proven effective in investigating local bone mineralization. Different research groups have used this technique to perform quantitative studies on the mineralization profile of bone mainly on iliac crest or proximal femur.(9–12) In addition, BSE could also be used to study other aspects of the cancellous bone remodeling. After its production by osteoblasts, the osteoid matrix undergoes a fast (primary) mineralization followed by a by a period of maturation during which mineral is gradually deposited over years (secondary mineralization). As a direct consequence of the progressive and quite slow secondary mineralization, each local wave of remodeling (or bone structural unit [BSU]) is easy to identify on BSE image (Fig. 1). While bone lamellation or cement lines have been used to assess the size of the BSU (to measure the wall thickness),(8) the gray levels can be used in BSE. A given trabecula looks like a patchwork of gray levels. Recent BSUs are relatively less mineralized and appear as dark gray. Older BSUs are more mineralized and appear whiter.

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Figure FIG. 1.. BSE image of a typical crescent shaped BSU (arrows). Osteocytes lacunae are clearly visible as well as bone lamellation. Old bone matrix (center of the trabecula) has higher gray level and higher mineralization. The recently formed bone matrix is darker because secondary mineralization is still in process.

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In this study, we used BSE microscopy on a large scale, examining 264 samples from human vertebral cancellous bone. Looking at the precise location of recent BSUs within the trabecular network, our working hypothesis was that two-dimensional (2D) perforations are not irreversible.

MATERIALS AND METHODS

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

Samples preparation

Samples were obtained from nine autopsy subjects.(13–15) In each subject, three vertebral bodies were obtained, one lumbar (L4), one from the thoraco-lumbar junction (T12 or L1), and one from the lower thoracic spine (T9). Two cylindrical cores (one anterior, one posterior) were extracted from each thoracic vertebral body, and three were extracted in the thoraco-lumbar or lumbar vertebral body because an additional external core could be obtained. This lead to a total of 68 cylindrical cores. These were fixed and embedded in spurr resin using a standard protocol.(16) The plastic blocks containing the whole cylindrical specimen were then cut at four levels (from bottom to top) using an Exact cutting grinding machine and re-embedded to obtain 268 blocks. The same blocks were polished with a 1-μm diamond finish and carbon coated. Four blocks, broken during the polishing, were not used.

BSE image collection

We used a XL30 SEM (Philips/FEI, Eindhoven, The Netherlands) with a BSE detector (Philips/FEI). The signal was first calibrated using C (Z = 6) and Al (Z = 13), and then the settings were changed to increase the contrast of the bone signal (Z ± 10). During the acquisition session, we controlled the drift of the signal using SiO2 as a standard (only for slight adjustments). Beam intensity was 20 kV.

Digital images were collected at ×100 magnification, each filed measuring 2 × 1.5 mm. Images were saved as TIFF files (645 × 484 pixels, pixel size = 3.21 μm). Nine adjacent images were obtained on each block (3 rows × 3 columns). Thirteen images were lost because of operator error.

Image analysis

A total of 2363 BSE images were examined for the two specific features described below. When a given feature could be identified on two adjacent images, it was only counted once.

We searched for microcalluses. A microcallus was defined as a globular structure containing woven bone (variable degree of mineralization, large osteocyte lacunae, pores for vessel).(5,17,18) Usually, the microcallus contained a mature trabecula or joined two trabeculae, although this was not a restrictive criteria.

We also looked for bridges. Here, a bridge is arbitrarily defined as follows (Fig. 2A): (1) a single BSU that connects two segments of trabeculae; (2) the BSU has a homogeneous gray level (generally containing osteocyte lacunae) that is lower than the connected elements; and (3) contrary to the edges of the connected segments of trabeculae, which usually show signs of osteoclastic resorption (irregular border and interruption of the cement lines), the surface of the bridge is smooth.

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Figure FIG. 2.. (A) Typical presentation of the feature arbitrarily defined as a bridge (Br) in this paper. It is a BSU that connects two segments of trabeculae (ST1 and ST2). These segments have been disconnected by osteoclastic resorption, as demonstrated by the scalloped border of the interruption of the cement lines. Oppositely, the external surface of the bridge is smooth showing no signs of resorption. (B) The arrows indicate how the length and breadth of the bridge were measured

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Microcalluses and bridges were identified and counted on each image. The length (μm) and the minimal breadth (μm) of each bridge was measured (Fig. 2B). In addition, we measured the bone surface and bone perimeter for each block, summing the values from nine images. Using simple macros under Qwin Pro (Leica Imaging System, Cambridge, UK), we computed the local trabecular thickness (Tb.Th, μm)(19) to compare this parameter with the breadth of the bridge.

RESULTS

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

Bridges were commonly observed in human vertebral cancellous bone. We found 396 bridges. Examples of bridges are shown in Fig. 3; some are very recent (Figs. 3G–3I), and some are older (more mineralized; Figs. 3D and 3E). Of 2363 images, there were 286 with one bridge, 48 with two bridges, and 5 with three bridges. By comparison, there were only 15 microcalluses (5 in subject 2, 2 in subject 3, and 4 in subjects 6 and 7).

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Figure FIG. 3.. Bridges (white arrows) were fairly easy to find in the vertebral trabeculae. (A) The first image represents the bridge that was fortuitously observed during a preliminary test of the BSE-SEM. This single observation triggered the present study. A 1.2 × 1.2 mm field is selected from the original documents that served for the measurements. Note that variety in breadth, length, and degree of mineralization. All bridges respond to the selection criteria detailed in the Materials and Methods section.

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Bridges were found in the cancellous bone of all the nine subjects included in this study (Fig. 4). Most of the subjects (7 of the 9) had 39 or more bridges. Only 16 bridges were found for subject 2 because the lumbar vertebra was excluded from the study (hence, 20 blocks were observed instead of 32).

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Figure FIG. 4.. Number of bridges for each of the nine subjects included in the study. Most subjects had 39 or more bridges. *For subject 2, only 20 blocks instead of the generally used 32 were examined, explaining the relatively low number of bridges. The relatively high absolute frequency of the bridge and the fact that they were found in all the subjects supports the idea that it is a common feature of normal bone remodeling of human vertebral trabecular bone.

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The size of the bridges varied from 29 to 869 μm, with a median value of 172 μm (Fig. 5). A total of 339 of the 396 bridges were longer than 100 μm, and 139 of the 396 were longer than 200 μm.

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Figure FIG. 5.. Histogram of the length of the bridges. Small bridges were commonly observed, but most bridges were longer than 100 μm. Dotted line is the mean trabecular thickness (85 μm).

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On comparing the Tb.Th with the breadth of the bridges for each subjects (Fig. 6), it appeared that for most of them, the mean breadth was 50–60 μm, which was significantly thinner than their respective Tb.Th. However, this was not systematic: subject 5 had quite thick bridges with a mean breadth of 83 μm, identical to the mean Tb.Th.

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Figure FIG. 6.. Comparison between the mean trabecular thickness (Tb.Th, filled circles) and the breadth of the bridges (open circles) for each subject included in the study. Error bar is ± SEM. For most subjects, the mean breadth of the bridge was smaller than the mean trabecular thickness. However, subjects 2 and 5 made bridges with a breadth identical to the calculated local trabecular thickness.

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DISCUSSION

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

On considering the data presented in this study, we propose reconsideration of the principle stating that perforations are irreversible.

Morphology and BSE

Boyde et al. showed few BSE images of vertebral cancellous bone, enhancing that the structural information contained in BSE images (namely microcallus or hypermineralized woven bone) should not be overlooked.(10) The present study confirms that BSE images can be used for other purposes than determination of mineralization profile. Here, it was used locate and delimit the BSU (Fig. 1). BSUs can be identified using other methods as well(7,8) (to measure the wall thickness), but with BSE microscopy, one can see which area in the trabecula is recent and which one is older. Restated, there is a direct access to the historical aspect of bone remodeling within the trabecula itself. Actually, when running a preliminary test on a few blocks, we observed an unexpected image of recent BSUs connecting two older segments of trabecula (this bridge is presented in Fig. 3A). This intriguing feature did not match the concept of irreversible perforation,(6) and consequently, we chose to investigate this issue on a large scale.

Normal remodeling?

The first issue regarding the interpretation of our data is whether it is either a rare, accidental, and bizarre observation or a normal feature of vertebral cancellous bone remodeling. An argument supporting the latter hypothesis is the absolute frequency of the bridge. In one of six images, we could see a bridge (396/2363). This rate should be compared with the much lower frequency of microcalluses (15/2363). It was also much easier to find a repaired perforation (a bridge) than a nonrepaired perforation (i.e., see Fig. 3J). Note that we were unable to quantify nonrepaired—or open—perforations because their identification is much too subjective.

Atypical observation could also result from the origin of the material. We studied autopsy specimens, and one or two subjects could have an unknown bone-related disease. However, we found bridges in all nine subjects. It is unlikely that all autopsy subjects were in that situation. Furthermore, we did not find any morphological arguments for bone disease in this investigation or in other studies on the same samples.(14,15)

Consequently, we propose that these images of BSUs connecting two segments of trabeculae is characteristic of normal remodeling in the human vertebral cancellous bone.

Perforations: the dogma

The classical view of bone remodeling cycle is that the osteoblasts refill the cavity created by the osteoclasts. Bone apposition happens in the concave surface of this cavity until partial or total refilling. When trabeculae are thicker than the depth of this cavity, the remodeling cycle leaves the architecture of the network unchanged. There is a substantial scatter in the depth of the cavities(2,20,21) and in the local thickness of a trabecula,(22) making the occurrence of fenestration or perforations likely to occur.(5) This is even more likely in the vertebral bone where the trabeculae are very thin. Now, what about bone apposition on the perforated trabecular plate? Accepting the principle that bone has to be deposited on a pre-existing bone surface, many authors have considered the perforation or fenestration of the trabeculae as irreversible.(2,20,22,), 23 Indeed, this was consistent with the progressive age-related loss of entire structural elements.(24) Overall, it is generally believed that large perforations (100–400 μm) are irreversible alterations of bone architecture.

Morphology of the bridges

The measured length of the bridges (the size of the repaired perforation) precisely covers that 100- to 400-μm range (Fig. 5), supporting the hypothesis that the BSUs defined as bridges in this study are repaired perforations. In addition, signs of osteoclastic resorption on both segments of reconnected trabeculae (Fig. 2A) were actually included in the definition of the bridge. On comparing the mean breadth of the bridges with the mean Tb.Th (Fig. 6), we found that, for most subjects, the repaired segment was thinner than the common trabecula. This means that either the thinnest trabeculae have been selectively perforated or that, for most subjects, the bridge is often thinner in its central portion. Note that we always measured the breadth as the minimal diameter (often in the middle portion of the bridge) and that some bridges were very thin because they were probably still in construction (low gray level, Figs. 3F–3I).

Osteoid bridges

If the mineralized bridges, as seen on BSE images, represent the new bone formed during a single wave of apposition, one should be able to find unmineralized bridges formed of only osteoid bone. In a previous study, we have reported the assessment of new bone formation indices on the same samples.(15) On reviewing these documents (trichrome Goldner-stained thin sections), we observed osteoid bridges (Fig. 7). The frequency of osteoid bridges (on the trichrome) was at least five times lower than that of mineralized bridges (BSE). This is normal, because mineralization of the osteoid takes place over few weeks, while mineralized BSUs probably remain for years (before being themselves remodeled).

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Figure FIG. 7.. Example of unmineralized bridge made of osteoid bone (trichrome Goldner thin section, bar = 100 μm). At this stage of bridge formation, the bridge could not be seen with the BSE microscope because it has the same density as the plastic resin. *An osteoclastic resorption cavity and **another osteoid seam are visible.

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Orientation of the lamellae

Generally, new bone is deposited with lamellae parallel to the eroded bone surface. In a perforated plate, new bone lamellae should be perpendicular to the big axis of the perforation (arrow for the length in Fig. 3B). However, on checking the bridges at high magnification or using the orientation of the osteocytes lacunae (Figs. 3 and 7), we found the lamellae axis to lie parallel to the big axis of the perforation.

Bridges and trabecular network

The problem of progressive disconnection of the trabecular network has been repeatedly described and illustrated on 2D documents.(25) Before the rise of three-dimensional (3D) morphology (with μCT for example), all connectivity parameters (Tb.N, Nd/Fe, Euler number, etc.) were obtained from 2D images. Each bridge reported in this paper is likely to increase the connectivity, as measured by these 2D parameters. For example, in Figs. 3A, 3G, 3H, 3I, 3K, and 3L, if there was no bridge, there would be two extra free-ends (Fe). In Figs. 3B-3F and 3J, no bridge would mean the loss of one node (Nd) and one extra Fe. Therefore, we can conclude that 2D disconnections seem to be reparable in human vertebral cancellous bone.

In 3D, interpretation is much more hazardous. Schematically, aggressive resorption on thin trabeculae may lead to two types of network “lesions”: perforations of the platelike elements and true disconnection of the thin (i.e., horizontal) bars.(5,26) Throughout this paper, we deliberately chose a reasonably conservative approach, considering the bridges as “reparation of perforations.” For the bridge to correspond to a true 3D reconnection (namely a bridge between two mechanically disconnected parts of an horizontal bar), it should be demonstrated that there was no bone above and below the level of the section, a point that will certainly require further investigation.

Conclusion

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

Whatever the 3D interpretation, it becomes difficult to consider the teams of osteoblasts as “cavity fillers” or “surface painters,” laboriously adding layers on an existing surface. They obviously have the capacity to lay down packets of bone that have a given shape and are going in a given direction; therefore, they should be considered as “active builders,” probably capable of protecting the trabecular network against excessive structural damage.

Acknowledgements

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

This work was supported by the National Funds for Scientific Research (Belgium).

REFERENCES

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