Micro-computed tomography
We previously showed that lyophilized collagen membranes possess osteoconductive properties in standardized calvarial defect models [15, 16]. However, the osteoconductive properties of native collagen membranes remained to be tested under these conditions. We covered 5-mm critical size defects in the left parietal bone with either regularly placed or upside-down membranes. Considering the bilayer membrane structure with an occlusive and a spongy layer, we hypothesized that the osteoconductive properties are affected by the membrane alignment. To this aim, we first assessed the bone coverage of the calvarial defect using μCT. Notably, both regular and upside-down membranes led to a virtually complete bone coverage of the defects (Figs. 2 and 3a).
Quantitative analysis showed that the ≈ 16 mm2 circular defect area inside the ROI was covered with new bone, amounting to 99.7% (96.0–100.0) in the upside-down group and 97.5% (85.6–100.0) in the regular group (p = 0.64) (Fig. 3b). We next calculated whether the overall volume of new bone was affected by the alignment of the collagen membranes. We found a tendency towards higher new bone volume in the upside-down group compared with the regular group, 8.0 mm3 (7.0–10.0) vs. 6.2 mm3 (4.3–9.4, p = 0.06). Consistently, mean trabecular thickness was significantly higher in the upside-down group compared with the regular group, 0.21 mm (0.19–0.23) vs. 0.18 mm (0.17–0.20, p = 0.03) (Fig. 3c, d). Despite this significant difference, these results imply that membranes placed upside-down can lead to similar bone regeneration compared with regularly placed membranes.
Histology
We next obtained thin-ground sections stained with Levai-Laczko dye to perform a descriptive histological analysis. Our approach allowed us to examine the three main tissue types of interest (bone with collagen, bone without collagen, and residual collagen fibrils) in the regular and upside-down groups (Figs. 4 and 5). We could not observe discernible differences between the two groups regarding the tissue areas and distribution patterns. We found a large portion of the new bone in the ectocranial area and a smaller portion in the central defect area. In both groups, a majority of new bone showed embedded collagen fibers. New bone with embedded collagen new bone was primarily found in the ectocranial area. New bone without collagen fibers was generally found in the central defect area, in close proximity to the defect edges or the dura mater. Between the periosteum and the new bone with collagen fibers, a discrete layer of collagen fibers was visible without new bone formation. Overall, descriptive histology revealed that both the evaluated areas and the spatial relationships of the different tissues were comparable between the regular and upside-down groups.
Histomorphometry, total bone
To further investigate these findings and quantify the various tissue areas within and around the defect, we performed a histomorphometric analysis. We differentiated three ROI (CD, ED, and EE) and six tissue types (bone with collagen fibers, bone without collagen fibers, soft tissue, mineralized fibers, residual collagen fibers, and brain prolapse) (Fig. 1). An overview of representative histological samples from the different groups is shown in Fig. 6a. Only the total new bone area tended to differ slightly between upside-down and regular groups, 3.9 mm2 (2.7–5.4) vs. 3.8 mm2 (2.2–4.0, p = 0.31) (Fig. 6b). Most of the new bone area, 75% in the upside-down and 80% in the regular group, was found in the ectocranial (ED and EE) ROI. In these ROI, we could also observe largely similar new bone areas in the upside-down and regular groups, 3.4 mm2 (1.7–4.6) vs. 2.6 mm2 (2.0–3.2, p = 0.40) (Fig. 6b). While total, ED, and EE new bone area all tended to be higher in the upside-down compared with the regular group, the differences were not significant. These results suggest that membranes placed upside-down lead to similar degrees of bone regeneration compared with regular membrane alignment.
Histomorphometry, bone with and without collagen
Next, we took advantage of the visualization of the remaining collagen fibrils now entombed in the new bone to examine whether the changes in bone formation are linked to the presence of collagen fibrils. We first measured overall new bone area with collagen fibers in all ROI. New bone area with collagen was comparable between upside-down and regular groups, 1.7 mm2 (1.2–3.5) vs. 1.7 mm2 (0.9–2.1, p = 0.39), respectively (Fig. 6c). Focusing on ED and EE, we could again observe comparable degrees of bone formation between upside-down and regular groups, 1.5 mm2 (1.1–3.3) vs. 1.5 mm2 (0.9–1.9, p = 0.44) (Fig. 6c). We observed virtually no bone with collagen fibers in CD. Moreover, there were no discernible differences related to collagen membrane alignment in bone without visible collagen fibers (Fig. 6d). Taken together, these findings suggest that treating calvarial defects with membranes placed upside-down leads to a highly comparable distribution of new bone tissue compared with regular membrane alignment.
Histomorphometry, mineralized collagen fibers
Previously, we identified regions with collagen fibers staining positive for mineralization but no visible bone-forming osteoblasts [15]. Here, we found similar areas of mineralized collagen fibers, mostly located in the ectocranial regions, between the new bone with collagen fibers and the periosteum. Upside-down and regular groups showed no differences with regard to the areas occupied by mineralized collagen fibers (Fig. 6e). At least in a rat calvarial model, collagen fibers appear to undergo mineralization. These findings suggest that the processes of collagen mineralization occur regardless of membrane alignment.