Alterations of mass density and 3D osteocyte lacunar properties in bisphosphonate-related osteonecrotic human jaw bone, a synchrotron µCT study

Abstract

Osteonecrosis of the jaw, in association with bisphosphonates (BRONJ) used for treating osteoporosis or cancer, is a severe and most often irreversible side effect whose underlying pathophysiological mechanisms remain largely unknown. Osteocytes are involved in bone remodeling and mineralization where they orchestrate the delicate equilibrium between osteoclast and osteoblast activity and through the active process called osteocytic osteolysis. Here, we hypothesized that (i) changes of the mineralized tissue matrix play a substantial role in the pathogenesis of BRONJ, and (ii) the osteocyte lacunar morphology is altered in BRONJ. Synchrotron µCT with phase contrast is an appropriate tool for assessing both the 3D morphology of the osteocyte lacunae and the bone matrix mass density. Here, we used this technique to investigate the mass density distribution and 3D osteocyte lacunar properties at the sub-micrometer scale in human bone samples from the jaw, femur and tibia. First, we compared healthy human jaw bone to human tibia and femur in order to assess the specific differences and address potential explanations of why the jaw bone is exclusively targeted by the necrosis as a side effect of BP treatment. Second, we investigated the differences between BRONJ and control jaw bone samples to detect potential differences which could aid an improved understanding of the course of BRONJ. We found that the apparent mass density of jaw bone was significantly smaller compared to that of tibia, consistent with a higher bone turnover in the jaw bone. The variance of the lacunar volume distribution was significantly different depending on the anatomical site. The comparison between BRONJ and control jaw specimens revealed no significant increase in mineralization after BP. We found a significant decrease in osteocyte-lacunar density in the BRONJ group compared to the control jaw. Interestingly, the osteocyte-lacunar volume distribution was not altered after BP treatment.

Figure 1. Slides of a reconstructed volume corresponding to a control jaw specimen are shown in A (x-y-plane) and B (x-z-plane).

The white dashed lines in A and B indicate where A is located in B and vice versa. C shows a minimum intensity projection, the projection range is 30 pixels (10.5 µm). D shows the lacunae mask corresponding to C, also in the form of an intensity projection (z-range = 30 pixels). Color bar in mass density of a.u.

Figure 2. In (A) a relative mass density distribution is shown.

The peak at around −100 is due to the air outside the sample, and the broad peak at about 200 is due to the glue used to attach the sample that managed to travel up the sample. In (B) the after segmentation is shown, and the parameters derived from it are indicated.

Figure 3. Volume renderings of two subsections originating from the jaw (A, B) and the femur (C, D. A and C show the lacunae and canals from a “top-down” perspective while B and D are shown from a “front-on” perspective.

The volumes of the lacunae are color-coded in units of µm³.

Figure 4. The are shown for the three regions, with region 1, region 2, and region 3 being the top, the middle, and the lower sub-section of one specimen scanned, respectively.

Figure 5. Investigated differences with respect to anatomical sites, assessed by analyses of variance (ANOVA), followed by post hoc multiple comparison Bonferroni tests are summarized.

Significant differences between groups are indicated by a horizontal bar. If the significance level was reached, the p-Value and the F-Value are reported.

Figure 6. Histograms of the lacunar volumes for the three different sites are shown.

Histograms are normalized to the area under the total number of lacunae for each site. Bin size is set to 50 µm³. The transparent areas indicate the standard error for each site based on the individual samples.

Figure 7. Histograms of all jaw lacunae grouped in either BRONJ or healthy bone.

The shaded areas correspond to the standard error based on the different samples. Histograms are normalized to the absolute amount of lacunae, bin size is 50 µm³. It should be noted that even though the histograms of the two groups look very similar, there are differences between the histograms of individual donors.