Introduction
Lactation is associated with large shifts in hormonal status. PTHrP and prolactin go up, PTH and estrogen go down [
1]. These hormonal changes have profound effects on osteoblasts, osteoclasts, and osteocyte activity. Bone remodeling is controlled by osteocytes in response to chemical, but for an important part mechanical, stimuli. Osteocytes sense mechanical signals placed upon bone (mechanosensation), transduce the mechanical signal into a chemical response (mechanotransduction), and consequently orchestrate the activity and recruitment of osteoblasts and/or osteoclasts by producing a multitude of signaling molecules [
2,
3]. It is likely that hormonal changes during lactation affect osteocyte response to mechanical signals as evidence exists that estrogen receptor-α (ERα), which is regulated by estrogen, contributes to the response of bone cells to mechanical strain [
4‐
6] and the PTH type 1 receptor has been suggested to be an important component of mechanical signal transduction in osteocytic MLO-Y4 cells [
7]. The sharp increase in PTHrP leads to a rapid expansion of the osteocyte lacunae, through a process called osteocytic osteolysis.
Osteocytes reside in cavities within the bone matrix called lacunae and interconnect through cell extensions (50–60 per cell) that reside in small canals named canaliculi [
2]. Through this extensive communication network, the osteocytes sense mechanical loads upon bone. Bone matrix deformations due to mechanical loading drive an interstitial fluid flow through the canaliculi. It has been suggested that the fluid flow activates osteocytes by generating streaming potentials [
8,
9], shear stress on the cell membrane [
10,
11], or by creating tension along the elements attaching the cell process to the lacunar wall [
12]. In addition, the inhomogeneities in the bone microstructure due to the osteocyte lacunar network can, in theory, locally amplify the matrix strain to a magnitude that is sufficient to directly activate the osteocyte cell bodies [
13], thereby eliciting a biological response [
3]. Building further upon these theories, the ability for osteocytes to sense mechanical stimuli placed on bone would be affected by canalicular morphology and by lacunar shape, since the latter could affect the strain amplification around the cell body [
14,
15]. For example, osteocytes residing in larger lacunae could experience higher strains. As a result, osteocytes would experience a locally modified mechanical environment, possibly resulting in an altered adaptive response to mechanical loading [
16]. There are data to support this hypothesis: It has been shown that the shape of osteocytes and their lacuna differs between several bone diseases; e.g., their shapes are significantly different in the tibia of individuals with osteopetrosis, osteopenia, and osteoarthritis [
17]. Furthermore, evidence suggests that the osteocyte lacunae are becoming smaller and more spherical with aging [
18,
19], while it has been shown that aging is associated with a reduced mechanoresponse [
20‐
22], which suggest that altered lacunar shape is a possible cause of impaired loading-induced bone remodeling process in the elderly. Yet, up to this date, experimental evidence supporting, or refuting, the presupposition that changes in lacunar morphology as seen in lactation, aging, and disease, can underlie an altered bone responsiveness to mechanical loads is non-existent.
The aim of this study was to quantify the response of osteocytes in mechanically loaded fibulae from lactating and virgin mice of similar age, gender, and genetic background, having different lacunar morphology. Because lactation is associated with altered hormonal status, we mechanically loaded excised fibulae rather than bones in vivo, in order to equalize hormonal conditions around osteocytes for at least 24 h before applying the mechanical stimulus. We hypothesized that osteocytes in bones from lactating mice show a stronger response to the mechanical loading, measured as higher loading-related changes in sclerostin expression as well as higher protein expression of β-catenin in comparison to osteocytes from virgin mice. We chose to measure β-catenin and sclerostin as parameters for the response to loading because these are well established to alter in expression in osteocytes after loading of bone [
23‐
25]. Considering the crucial role of osteocyte to maintain healthy bone, a better understanding of the way osteocyte shape is related to its capability to direct bone formation and resorption may help to unravel the origins of reduced bone adaptive response as seen with, for example, lactation, disease, and aging.
Discussion
In this study, we aimed to quantify the response of osteocytes in mechanically loaded fibulae from lactating and virgin mice of similar age, gender, and genetic background, having different lacunar morphology, as measured by quantifying protein expression of the established mechanoresponsive proteins sclerostin and β-catenin as well as Sost gene expression. We found a stronger loading-induced reduction in sclerostin and stronger increase in β-catenin expression by osteocytes residing in the fibulae of lactating mice as compared to the response in virgin mice. Mechanical loading significantly affected Sost expression in lactating mice not in virgin mice, indicating that osteocytes in fibulae from lactating mice may respond better to mechanical loading. Hence, our experimental findings support the idea that lacunar morphology affects osteocyte signaling in situ.
In order to investigate the effect of lacunar morphology on the osteocyte mechanoresponse, we chose lactating mice wherein the osteocytes enlarge their lacuna volume by removing bone from their perilacunar bone matrix in a process called osteocytic osteolysis, in response to increased demand for calcium during lactation [
26,
27]. Utilizing nano-CT to quantify lacunar morphology, the present study further supports previous findings of lactation-induced osteocytic osteolysis [
27] and demonstrated significantly larger lacunae in the fibulae of lactating mice as compared to virgin mice.
For this study, we took advantage of murine fibulae as their small size facilitated analysis by Nano-CT since a single field of view encompassed the entire bone’s cross section; hence no cutting nor preparation was required. Furthermore, the fibula is a load-bearing bone that adapts to mechanical loading; indeed, several groups have successfully used murine fibulae to study skeletal mechanobiology and demonstrated bone adaptation to mechanical loading in a similar way as in the tibia [
36‐
39]. In addition, the fibula is small enough to allow osteocytes to survive in their matrix for up to several days in the absence of blood flow. Accessing the viability of human osteocytes in bone chips, similar in size to our mouse fibulae, as quantified by live-dead staining as well as expression of osteocyte-specific markers shows that 60% of the osteocytes are alive and functional after 7 days of culture [
40]. This indicates that osteocytes in their native matrix can remain alive up to 7 days, which is much longer than the 48 h after excision of the fibulae when our experiments were terminated.
A limitation of using the lactating mouse model is the obvious hormonal differences between lactating and virgin mice. The most important differences are the higher PTHrP and prolactin levels in lactating mice. In order to diminish the effect of hormones in lactating group, all fibulae were isolated from the body and incubated in culture medium without hormones 24 h before applying ex vivo mechanical loading to equalize conditions before and during the application of the mechanical stimulus. Through this procedure, the direct effect of hormones has been limited, but are unlikely to be completely nullified. There is accumulating evidence that estrogen receptor-α (ERα), which is regulated by estrogen, contributes to the response of bone cells to mechanical strain [
4‐
6]. Pregnancy is associated with high estrogen levels, the major hormonal regulator of bone metabolism [
4,
27]. Estrogen levels then rapidly (within a day) return to baseline levels post partum. It has been suggested that dropping estrogen levels cause a change in setpoint of the mechanostat, rendering osteocytes
less responsive to mechanical stimuli. A drop in estrogen levels would likely decrease osteocyte mechanosensitivity in lactating mice, which is opposite to our findings. Thus, it is unlikely that changes in estrogen levels explain our results. Little to nothing is known regarding the effect of prolactin on the osteocyte mechanoresponse. The PTH type 1 receptor on the other hand has been suggested to be an important component of mechanical signal transduction in osteocytic MLO-Y4 cells [
7], but PTHrP and mechanical loading restore bone mass and strength in a diabetic mouse in an additive manner rather than a synergistic manner [
41], suggesting that PTHrP does not alter the inherent response of osteocytes to mechanical stimuli. This makes it even less likely that altered PTHrP levels explain our results. Lytic enzymes that are upregulated in osteocytes exposed to hormones during lactation include TRAcP, cathepsin K, and MMP13. MMP14 has been shown to affect the response of osteocytes to mechanical loading in vitro [
42], suggesting that certain types of MMPs may affect the osteocyte mechanoresponse. However, in order to affect the mechanoresponse, molecules need to be in a molecular favorable position, such as the transmembrane proteins integrins. MMP14 is such a transmembrane protein, while TRAcP, cathepsin K, and MMP13 are not. Together, these data make it likely that the enhanced response of osteocytes in fibulae of lactating mice is due to the enlarged lacunae rather than the direct effect of altered hormone levels on the osteocyte mechanoresponse. Further studies need to investigate whether expression of genes activated by prolactin and PTHrP persisted in the osteocytes of lactating mice, in order to exclude the possibility that these hormones enhance the mechanical response, separate from changing the lacuna morphology.
Using fibulae has some limitations. The very small size of tiny mouse fibulae leads to difficulties with paraffin sectioning resulting in relatively low number of good sections. Furthermore, the straight alignment of fibula during gluing the sample in the bone holder is important as a skewed position would affect the strain distribution in the fibula during compression loading and therefore the osteocyte response to mechanical loading. This is difficult to achieve with a small and fragile bone such as the fibula. We discarded any samples which were not perfectly aligned, thereby ensuring validity of our experiments. These two technical issues caused the unequal sample sizes between the groups. In addition, in this study, we focused our quantification of lacuna size at the distal part of the fibula and we do not know whether lactation also affects the size of lacunae in the middle and proximal parts of fibulae. Since Sost gene expression was measured in osteocytes residing through the whole fibula, it is possible that a lack of difference in lacuna size in the proximal side of the fibulae between lactating and virgin mice caused the non-significant difference in the magnitude of Sost response to mechanical loading between lactating and virgin mice.
Finally, a limitation of this study is that we have used antibodies for visualization of β-catenin that do not discriminate between the phosphorylated and unphosphorylated form. However, it has been well established that mechanical loading in bone leads to inhibition of GSK-3β, via phosphorylation of AKT, initiated by factors such as nitric oxide, focal adhesion kinase, and prostaglandin E
2 in response to mechanical stimuli [
43‐
46]. As a result of GSK-3β inhibition, β-catenin stays unphosphorylated upon a mechanical stimulus, is no longer targeted for degradation, accumulates inside the mechanically stimulated osteoblast or osteocyte, and finally initiates gene transcription. Increases in β-catenin protein expression in these cases are thus not to be attributed to changes in mRNA expression. Therefore, we chose to measure β-catenin only at the protein level. Production of canonical Wnts by mechanically loaded cells could in theory also contribute to inhibition of GSK-3β in osteocytes, and subsequent accumulation of β-catenin, although a study by Lara-Castillo et al. [
43] suggests that Wnts play no role in the increase in β-catenin as observed in osteocytes in vivo in response to loading. Increases in β-catenin levels as observed in response to mechanical stimuli can thus almost certainly be attributed to accumulation of the unphosphorylated (active) form.
Using micro-finite element modeling, we previously demonstrated that the lacunar geometrical features can affect the mechanical environment of the osteocytes, such that lacunae with larger volume experience higher maximum effective strains than lacuna with small volume. Osteocytes exposed to higher maximum effective strains respond stronger than those exposed to lower strains [
25], thus at the same overall load placed on bone, bones with larger lacunae are expected to elicit a stronger response in the osteocytes. This is in line with the experimental findings as obtained in the present study.
We previously showed that the osteocyte lacunae are becoming smaller and more spherical with aging [
19,
28]. The findings of the present study indicate that bones with smaller lacunae respond less to mechanical loading than bones with larger lacunae, suggesting that smaller lacunae in aged bones are a potential causative factor for the reduced bone mechanoresponsiveness with aging. Our findings may also explain the slower orthodontic tooth movement in elderly as compared to young people. Orthodontic tooth movement is a biological consequence of alveolar bone remodeling induced by mechanical force and controlled by osteocytes [
47]. Previous studies have shown that parathyroid hormone treatment [
48,
49], lactation, and chronic dietary deficiency of calcium [
50] could accelerate the rate of tooth movement through enhancement of alveolar bone remodeling process. We are well aware that all of these interventions potentially directly affect osteocytes, osteoblasts, or osteoclasts, so no strong conclusions with regard to osteocyte mechanosensing are drawn. Yet, all of these factors are also associated with osteocytic osteolysis [
26] and enlargement of the lacunar network. Thus, these findings, at the very least, do not oppose our hypothesis that osteocytes residing in enlarged lacunae show a stronger mechanoresponse.
In summary, we found that the osteocytes in fibulae of lactating mice, which are embedded in larger lacunae, show a greater response to mechanical loading in comparison with the osteocytes residing in fibulae of virgin mice. Our results support the theory that osteocyte lacunar morphology affects bone mechanotransduction. Given the ability of osteocytes to orchestrate osteoblast and osteoclast behavior, and their ability to shape their immediate microenvironment, a better understanding of the relationship between osteocyte lacunar shape and bone mechanobiological response may reveal whether altered lacunar shape is one of the pathways involved in lactation, disease, and age-related bone loss.