Bone remodeling in response to fatigue-induced microdamage is a spatially constrained process where focal tissue injury leads to localized osteocyte apoptosis followed by targeted resorption of the damaged region and its dying cells [18
]. Osteocyte apoptosis also plays an obligatory role in initiating the remodeling response to damage [4
], but it is not known if apoptotic osteocytes themselves produce the signals needed to stimulate osteoclast differentiation. The results of this study show that dying and signaling osteocytes are discrete populations. While dying osteocytes did not produce pro-osteoclastogenic signals, a population of non-apoptotic osteocytes surrounding the apoptotic cells near the microdamage zone upregulated expression of RANKL and VEGF and decreased expression of OPG in a spatially and temporally coherent fashion.
The ability of cells within the osteoblast lineage to stimulate osteoclast formation and/or activity was proposed by Rodan and Martin in 1981 [38
], but only recently has it become clear that this capacity was maintained and probably enhanced as osteoblasts mature into osteocytes [14
]. Strikingly, selective ablation of RANKL expression in the osteocyte population in mice led to reduced resorption and its phenotypic consequences during development, and an impaired ability to respond to mechanical unloading [14
]. The fatigue microdamage repair model seems particularly well suited to the examination of pro-osteoclastogenic signaling by osteocytes for two reasons. First, baseline intracortical remodeling is zero in this model [19
]. This appears to differ from some of the mouse studies [14
], which were carried out in younger animals where bone resorption rates are higher and more RANKL expression is observed. Consistent with the absence of resorption, we found osteocyte RANKL expression to be negligible by IHC while OPG was constitutively expressed. Second, unlike development and hindlimb unloading, induction of bone remodeling in response to microdamage is highly localized, and allows the relationship between pro-osteoclastogenic signaling to be assessed with respect to both the inducing stimuli (damage and the requisite osteocyte apoptosis) and response (subsequent remodeling). Our findings that a dramatic induction of an increased RANKL/OPG ratio occurred after damage, was limited to a finite region where resorption consistently occurs in this model, and yet extended far enough to reach the periosteal surface where remodeling initiates, strongly support a direct role for osteocytes – specifically non-apoptotic osteocytes – in RANKL based signaling to osteoclast progenitors.
The distribution of osteocytes showing pro-osteoclastogenic signaling characteristics effectively excluded the apoptotic cells, and corresponded to the distribution of viable osteocytes that surrounded fatigue damage and were shown by Verborgt et al. [18
] to actively protect themselves from death by upregulating the anti-apoptotic protein Bcl-2. This similarity includes comparable patterns of decline in Bcl-2 expression and pro-osteoclastogenic cytokine expression with increasing distance from microdamage. Whether the population of signaling osteocytes that we observed overlaps fully or partly with the Bcl-2+
population is as yet unclear, but this question can be investigated in future using the double staining approach used in the present study. Also of interest, the distance spanned by the signaling osteocytes relative to the microdamage site (150–200 µm emanating from the damage site) would be sufficient to permit the osteocytederived pro-osteoclastogenic signal to reach the periosteum and its blood supply containing osteoclast progenitors. Finally, the proapoptotic signal pattern was sustained for at least 7 days post-injury, adequate time for the differentiation of osteoclasts.
The distributions of RANKL and OPG expression following fatigue indicated a shift in the signaling environment surrounding the microdamage area to one favoring osteoclast formation, indicated by upregulation of RANKL and corresponding downregulation of OPG. RANKL is typically formed as a membrane bound molecule (40–45 kDa) which under the appropriate conditions can be cleaved by metalloproteinases into a smaller soluble form(30 kDa) [41
]. Immunohistochemical staining cannot distinguish between membrane bound and soluble RANKL in this model system, the available antibodies that work in rodent tissues bind to epitopes that are common to both forms. However, it seems reasonable to hypothesize that osteocyte signaling involves both the membrane bound and soluble forms. The molecular size order for soluble RANKL would allow it to move readily easily through the osteocyte lacunar-canalicular system [12
]. That up-regulation of RANKL staining in osteocytes is highest some 150–200 µm from the microdamageosteocyte apoptotic core would place this signaling in direct proximity to the nearest bone surface (periosteal or endocortical) at which osteoclast precursors can be recruited. Likewise, OPG, which was found consistently in osteocytes, is a soluble factor that has a molecular size of approximately 60 kDa and should also readily pass through the osteocyte lacunar-canalicular network.
M-CSF has been widely shown to be a necessary factor, along with RANKL, for osteoclastogenesis [42
]. Surprisingly, we found no change in M-CSF mRNA expression in fatigue-loaded ulnar diaphyses and confirmed this finding in several independent replications of the experiments and analyses. These data show that osteocytes in fatigued bone do not upregulate production of this key regulatory molecule during the osteoclast activation phase in this model. It is possible that the levels of M-CSF expressed constitutively are adequate for osteoclast differentiation following fatigue loading, or that upregulation does occur in a small subpopulation of cells which cannot be detected by these methods. Furthermore, the unique anatomy of the ulnar mid-diaphysis which, as noted earlier, has effectively no marrow cavity, may also play a role. Since M-CSF is produced by marrow stromal cells [45
], the lack of detectable change in M-CSF expression may be due to the lack of a significant marrow compartment in the rat ulnar diaphysis.
The current experiment revealed that VEGF staining in osteocytes was spatially similar to RANKL, but temporally different. Osteocyte expression of RANKL was significantly increased at 3 days after fatigue, while VEGF production reached significance only at 7 days. VEGF may potentially play several different roles in this system. Expression for this factor is linked to the stabilization of Bcl-2 via the MAP kinase cascade and could contribute to the increased Bcl-2 expression by osteocytes seen by Verborgt et al. as an anti-apoptotic response to microdamage [18
]. Previous studies have also shown VEGF to be involved in the regulation of osteoclastogenesis, both directly and upstream of RANKL [49
]. However, the late increase in VEGF compared to RANKL appears most consistent with a role in regulating angiogenesis – also an essential element of bone remodeling – and suggests that osteocytes may play a central role in the coordination of this process as well. Since the current studies were limited to times before the appearance of new remodeling spaces and their neovascular elements in this model, future longer term studies will be needed to directly test this hypothesis.
In summary, our results demonstrate clear spatial and temporal relationships between injury, osteocyte apoptosis and pro-osteoclastogenic signaling in response to fatigue-induced microdamage. This represents the first demonstration, to our knowledge, of pro-osteoclastogenic signaling by osteocytes in the context of a targeted bone remodeling process. These results provide evidence that RANKL expression by osteocytes is low or absent in adult cortical bone osteocytes but is inducible by injury. The finding that apoptotic cells are essential to the initiation of bone remodeling but do not themselves carry out RANKL-based pro-osteoclastogenic signaling indicates a division of labor among osteocytes in response to damage paralleling that seen in ischemic injury. Such similarities suggest that common pathways and mechanisms probably exist in localized remodeling of many tissue types.