Fracture healing is a complex process that takes place in order to fully restore anatomic and functional structure following injury, involving cell proliferation and differentiation, chemotaxis, and synthesis of extracellular matrix (ECM). Although delayed healing of femoral neck fractures in OP patients has been reported [
5], no studies reporting impaired healing in OP compared to healthy age-matched controls have been published. Due to ethical concerns and multiple confounding factors, most studies have been performed in animal models. The ovariectomized (OVX) rat model has been approved by the FDA [
6] as a relevant model for the study of postmenopausal OP, mimicking postmenopausal trabecular bone loss when examined over relatively short periods of time. Impaired fracture healing has been reported in this model in early [
7] and late [
8] stages, as evaluated by radiographic, histomorphometric, and mechanical parameters. However, we recently demonstrated preserved bone mineral density (BMD) and mechanical strength of callus 6 weeks after fracture in experimental OP [
9], in line with the findings by Kubo et al. [
8] at the same time point. In support, Wheeler et al. [
10] report no difference between OVX and sham rats in mechanical strength of callus at 4, 6, and 8 weeks after fracture. Also, recent data support unaltered fracture healing mechanisms during OP [
11]. Thus, data are conflicting and, moreover, only a limited number of studies look into the molecular events involved in fracture repair in OP.
An additional predictor in the development of postmenopausal OP is vitamin D deficiency, which also may influence bone repair. Vitamin D deficiency is common among women with OP [
12] and women with fractures regardless of age [
13], leading to accelerated bone resorption. In support, OVX rats with vitamin D depletion develop site-specific bone loss similar to what is observed in women with postmenopausal OP [
14]. Additionally, oral administration of 1,25(OH)
2 vitamin D
3 has recently been shown to improve fracture healing in OVX rats [
15]. Interestingly, both estrogen [
16] and vitamin D [
17,
18] influence the expression and synthesis of ECM proteins, and such proteins play important roles in mediating cellular function and may serve as important modulators of bone regeneration.
Thus, we hypothesized that lack of estrogen and vitamin D will influence global gene expression as well as the synthesis and ultrastructural distribution of ECM molecules in the fracture callus and, consequently, the capacity of fracture repair. To test this hypothesis, we examined global gene expression and in situ mRNA expression as well as the ultrastructural protein distribution of two major phosphoproteins in bone, i.e., osteopontin (OPN) and bone sialoprotein (BSP), and two osteoclast enzymes, i.e., tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTK), in the callus 3 and 6 weeks after tibial fracture of vitamin D-depleted OVX rats. Sham-operated, age-matched controls were used for comparison.
Discussion
The present study addresses fracture healing in vitamin D-depleted OVX rats at the molecular level over a 6-week period. This is a well-established model, and in a recent study we observed bone loss in femoral neck and lumbal vertebrae but, somewhat surprisingly, little effect on the mechanical strength of healing fractures [
9]. Estrogen and vitamin D exert their effects on bone in a spatial manner, which differs between metabolically active trabecular bone and weight-bearing cortical bone. Although vitamin D initially was reported to stimulate bone resorption in vitro [
29], more recent in vivo findings indicate that active vitamin D analogues rather have an inhibitory effect on bone resorption, at least in a state of high turnover [
30]. This inhibitory effect is reported to be less active in trabecular bone [
31]. In a similar manner, estrogen deficiency following OVX results in bone loss at discrete sites in the skeleton, with most pronounced effect on trabecular bone [
32].
Several genes associated with signal transduction, lipid and protein metabolism, ionic and protein transport, and neuropeptide and G-protein signaling were differently regulated in our OVX-D rat model. Many of these processes represent metabolic systems capable of maintaining bone homeostasis and tissue turnover. Most genes were expressed in an increased fashion in OVX-D compared to sham, in line with other reports on gene profiling of intact bone in OP [
33]. Interestingly, no significant differences were detected among genes encoding noncollagenous proteins (NCPs) with known functions in bone metabolism.
At 3 weeks, sevenfold more genes were differently regulated compared to 6 weeks; and consequently, changes in the healing pattern in OP would be expected in early stages. Among the differently regulated genes, the genes for IL-17 receptor A and FGF23 were up- and downregulated in OVX-D after 3 weeks. FGF23, a phosphaturic hormone and part of the newly discovered hormonal bone–parathyroid–kidney axis, is modulated by PTH, vitamin D, and phosphate levels. The fact that 1,25(OH)
2D
3 upregulates FGF23 gene expression in bone [
34] and undetectable serum levels of FGF23 are found in mice lacking the vitamin D receptor [
35], downregulation of FGF23 in the OVX-D group could be explained by lack of dietary vitamin D. However, downregulation due to vitamin D deficiency would be expected to be present also after 6 weeks. Interestingly, FGF23 has recently been suggested as a putative marker of bone healing; decreased FGF23 mRNA expression is associated with delayed fracture healing in ovine bone [
36]. Thus, downregulation of FGF23 in our model could also be associated with a possible delay in fracture healing at an early stage, although this is not supported by our other findings. IL-17 plays a role in bone loss in rheumatoid arthritis [
37]. However, IL17RA null mice exhibit more pronounced bone loss compared to wild-type mice following OVX [
38]; and thus, the role of this interleukin and its receptor seems diverse. Our observations call for further studies on the role of FGF23 and IL17RA during fracture healing in osteoporotic bone.
In our molecular studies we focused on four molecules with roles in tissue turnover taking place in the callus and, putatively, influenced by lack of estrogen and vitamin D depletion. For example, OPN is suggested to play roles both during osteoclast development/activity [
39] and during matrix mineralization [
40]. Furthermore, OPN null mice are resistant to OVX-induced bone loss [
41] and display disturbed fracture healing [
42], suggesting a role for OPN during estrogen deficiency-related bone loss as well as in bone repair. Moreover, OPN is under the influence of both estrogen and vitamin D [
16,
18].
mRNA expression for OPN, BSP, TRAP, and CTK in cell types was distributed as previously described in intact bone tissue [
21,
26,
43]. OVX-D animals presented an increased ratio of OPN mRNA-expressing cells and a decreased ratio of BSP mRNA-expressing cells at 3 weeks and a significantly reduced ratio for BSP after 6 weeks of healing. These observations are in line with the reported inverse regulatory role of vitamin D for these proteins in vivo [
44]. The observed increase in OPN and subsequent decline in BSP could possibly indicate suppressed osteoblastic differentiation in the OVX-D group.
In OVX-D rats at 3 weeks relative to 6 weeks, there was a significant decline in the ratio of cells expressing mRNA for OPN, BSP, and CTK but not that for TRAP. Thus, the decreased number of cells producing these bone turnover biomarkers in the OVX-D group at 6 vs. 3 weeks indicates a decline in synthetic activity/bone remodeling and a more remodeled callus. The latter supports the finding of Cao et al. [
45] after 16 weeks of healing of a histologically more mature callus with preserved mechanical properties in OP vs. sham rats. Furthermore, these authors report no differences in healing after 6 weeks, in line with our results.
Quantitative immunoelectron microscopy showed only minor differences in labeling (Tables
5,
6,
7). The apparent inconsistency between our ISH and immunogold data is most likely due to the fact that the two data sets represent different aspects of callus turnover: while ISH data represent mRNA expression and thus protein synthesis, immunogold data reflect the present amount of protein in the tissue, which is the net result of synthesis, secretion, and degradation. That only minor differences of protein synthesis and tissue distribution were detected between the groups is supported by other studies [
46,
47]. Interestingly, also recent studies of gross mechanisms in fracture healing in OVX rats [
11] and transgenic mice [
48] support our findings.
As with any animal model, there are limitations in simulating clinically relevant conditions. The calluses showed considerable heterogeneity both by macroscopic appearance and by histology, which was also reflected by large standard deviations in histomorphometric and BMD measurements. Except for a significant difference in tissue composition after 3 weeks, where the OP group presented calluses comprising more bone and cartilage and less fibrous tissue compared to sham, there was no systematic difference between the groups with respect to Ob.S/Oc.S, BMD, or histomorphometric parameters. Mechanical instability during fracture healing promotes cellular differentiation in the direction of endochondral bone formation [
49], and variable degrees of fracture fixation in our experiment may explain the heterogeneity in the callus sample. It should be noted that since mechanical stress influences the mRNA expression in bone cells [
50], this may have influenced our results by causing a relatively large variation among the samples. Furthermore, minimal-trauma fractures in OP patients are mostly localized to the proximal femur, vertebral column, and distal forearm [
51], i.e., areas of high trabecular to cortical ratio. Thus, the present study concerns fracture healing in cortical bone, and it is possible that this type of bone healing is less influenced by estrogen and vitamin D deficiency.
In summary, several genes are differently regulated in OP induced by OVX and vitamin D depletion, mostly in an early stage and the majority in an increased fashion, although no genes encoding NCPs with known function in bone metabolism were differently expressed. Against the background of only minor differences in the synthesis and protein expression of OPN, BSP, TRAP, and CTK in callus between OVX-D and sham-treated animals, our results suggest that the molecular composition of the fracture callus is not markedly skewed in OP.