Slight comminution was reported for a high percentage of all usable closed femur fractures created according to Bonnarens et al. [
10]. This finding extensively interferes with correct segmentation and requires reliable, easy segmentation procedures. Therefore, we applied a fully automated method to identify and separate all parts of the callus, original cortical bone and marrow without requiring determination of specific ROIs. In addition, we assessed the non-volume-dependent μCT parameters for association with biomechanics.
Contrary to our findings, the ‘semi-automated’ image segmentation method, as described by Morgan et al. in 2009 and again in 2016, precludes the measurement of the endosteal callus. However, the latter contributes to the stability of the entire structure. Morgan et al. also remark that mechanical contributions occur at the level of the ‘entire’ callus [
5,
11]. Dickson et al. showed increased biomechanical stability with increased endosteal calcified tissue volume after femur fractures [
15].
Our study was not originally designed to provide a systematic correlation or comparison of methods; we instead aimed to assess the influence of medication on fracture healing (data not shown). However, when interpreting these data, uncertainty was apparent concerning the impact of various parameters. Therefore, we further evaluated the association between μCT and biomechanical data between different animals. Based on the different processing of the two methods, we could not compare the same femura for association of single values between μCT and biomechanics. However, we could compare the medians of the groups, resulting in fewer values with a minor impact. Nevertheless, conclusions can be drawn from the results. The weight range of our rats was very narrow such that results for femura from different rats are comparable [
13].
Unlike the reported positive correlations between BV and biomechanics of unfractured femura [
16] and the very strong positive correlations between BMC and the structural strength using pQCT and DXA (
r > 0.95) [
6,
17,
18], our results yielded a strongly negative correlation between the load and BV (
r = −0.75), BMC (
r = −0.73) or BS (
r = −0.70). However, the study approach for a regenerating callus is completely different from studies evaluating unfractured bone [
19]. In addition, the level of fracture comminution is positively correlated with the amount of callus [
10]. Nyman et al. previously showed an inverse correlation between μCT callus BV and strength (the body tries to compensate for the reduced stability) after 28 days, placing these results in a logical context [
9]. With regard to TMD, no relevant association could be seen between the measures (
r = −0.08). Shefelbine et al. reported a good predictive value of biomechanical measurements in osteotomy models (clear gap) for density measurements. However, in more complex fractures (fragmentation), which are frequently obtained by closed fracture mechanisms according to Bonnarens et al., density measurements lead to poor predictions of fracture callus biomechanics [
10,
19].
Again, in contrast to our initial expectation, the SMI had a strongly positive correlation with the breaking load (
r = 0.75). During ageing (osteoporosis) and disease, plates are perforated and connecting rods are dissolved as part of a transition from more stable (and dense) plate-like (SMI ≤0) to less stable rod-like (SMI = 3) trabeculae [
2,
20]. The SMI of the prednisolone group was considerably lower (−1.30) than the SMI of the other groups. In combination with the lowest load, the latter led to the positive association between the groups. Unlike the biomechanical evidence, the trabecular structure of the prednisolone-affected callus indicates there is a more stable callus. DA, representing the orientation of the trabecular structure responsible for the anisotropic properties of the bone, revealed a weakly negative correlation (−0.45). [
21]. Tb. Th. revealed a strongly negative correlation (
r = −0.70). The notable, but unexpectedly, lowest load (46.28) compared with the highest Tb. Th. (0.1004) in the prednisolone group contributed to the negative association between the groups. Qualitative evaluation of the colour-coded μCT reconstructions, representing the Tb. Th., showed slightly thicker trabecular-like struts in the prednisolone group compared with those of the other groups (Fig.
4d). Prednisolone calluses seemed to have a lower porosity (note the increased TMD) compared with the other groups. Around the 21st day, the cartilage and original cortical bone tissue are generally remodelled, increasing the void spaces between the built trabeculae [
22]. Histologically, a delayed healing of 5 days has previously been described after prednisolone medication, which might explain the thicker trabeculae and the increased thickness compared with that of the other groups [
23]. Additionally, the thicker (Tb. Th.) and plate-like (SMI) trabeculae, assumed to have higher stability, do not increase the stability because of the missing outer periosteal callus fracture gap-bridging that is typically observed within this group (Fig.
4c) [
22]. Consistent with our findings, increased outer bony bridging has previously been demonstrated to be able to enhance the correlation of μCT parameters with strength [
9]. Therefore, bridges across the fracture gap, particularly the outer periosteal callus, can be considered relevant for biomechanical properties, even if they only slightly contribute to the total callus. Our study has shown that the localisation of the callus is important for biomechanical properties; this might explain the unexpected correlations between several biomechanical and structural measures (BV, SMI and Tb. Th.) [
22,
24]. Therefore, therapies that enhance bone healing and lead to a bridging callus with increased load might lead to stronger, positive (vice versa for SMI) associations between the modalities and reach statistical significance [
8]. This implication is particularly important in a clinical sense because an increasing number of studies are assessing the influence of medication (e.g. rivaroxaban and tadalafil) on fracture healing and bone remodelling, respectively [
25,
26].