Oran D. Kennedy

Activation of resorption in fatigue-loaded bone involves both apoptosis and active pro-osteoclastogenic signaling by distinct osteocyte populations.

Osteocyte apoptosis is required to initiate osteoclastic bone resorption following fatigue-induced microdamage in vivo; however, it is unclear whether apoptotic osteocytes also produce the signals that induce osteoclast differentiation. We determined the spatial and temporal patterns of osteocyte apoptosis and expression of pro-osteoclastogenic signaling molecules in vivo. Ulnae from female Sprague-Dawley rats (16-18weeks old) were cyclically loaded to a single fatigue level, and tissues were analyzed 3 and 7days later (prior to the first appearance of osteoclasts). Expression of genes associated with osteoclastogenesis (RANKL, OPG, VEGF) and apoptosis (caspase-3) were assessed by qPCR using RNA isolated from 6mm segments of ulnar mid-diaphysis, with confirmation and spatial localization of gene expression performed by immunohistochemistry. A novel double staining immunohistochemistry method permitted simultaneous localization of apoptotic osteocytes and osteocytes expressing pro-osteoclastogenic signals relative to microdamage sites. Osteocyte staining for caspase-3 and osteoclast regulatory signals exhibited different spatial distributions, with apoptotic (caspase 3-positive) cells highest in the damage region and declining to control levels within several hundred microns of the microdamage focus. Cells expressing RANKL or VEGF peaked between 100 and 300μm from the damage site, then returned to control levels beyond this distance. Conversely, osteocytes in non-fatigued control bones expressed OPG. However, OPG staining was reduced markedly in osteocytes immediately surrounding microdamage. These results demonstrate that while osteocyte apoptosis triggers the bone remodeling response to microdamage, the neighboring non-apoptotic osteocytes are the major source of pro-osteoclastogenic signals. Moreover, both the apoptotic and osteoclast-signaling osteocyte populations are localized in a spatially and temporally restricted pattern consistent with the targeted nature of this remodeling response.

Osteocytes: Master Orchestrators of Bone

Osteocytes comprise the overwhelming majority of cells in bone and are its only true “permanent” resident cell population. In recent years, conceptual and technological advances on many fronts have helped to clarify the role osteocytes play in skeletal metabolism and the mechanisms they use to perform them. The osteocyte is now recognized as a major orchestrator of skeletal activity, capable of sensing and integrating mechanical and chemical signals from their environment to regulate both bone formation and resorption. Recent studies have established that the mechanisms osteocytes use to sense stimuli and regulate effector cells (e.g., osteoblasts and osteoclasts) are directly coupled to the environment they inhabit—entombed within the mineralized matrix of bone and connected to each other in multicellular networks. Communication within these networks is both direct (via cell–cell contacts at gap junctions) and indirect (via paracrine signaling by secreted signals). Moreover, the movement of paracrine signals is dependent on the movement of both solutes and fluid through the space immediately surrounding the osteocytes (i.e., the lacunar–canalicular system). Finally, recent studies have also shown that the regulatory capabilities of osteocytes extend beyond bone to include a role in the endocrine control of systemic phosphate metabolism. This review will discuss how a highly productive combination of experimental and theoretical approaches has managed to unearth these unique features of osteocytes and bring to light novel insights into the regulatory mechanisms operating in bone.

Osteocyte apoptosis is required for production of osteoclastogenic signals following bone fatigue in vivo.

Osteocyte apoptosis is spatially, temporally and functionally linked to the removal and replacement of microdamage in the bone. Recently we showed that microdamage elicits distinct responses in two populations of osteocytes near the injury site. Osteocytes directly adjacent to microdamage undergo apoptosis, whereas there is a second group of osteocytes located adjacent to the apoptotic population that upregulate expression of osteoclastogenic signaling molecules. In this study we used the pan-caspase inhibitor QVD to test the hypothesis that osteocyte apoptosis is an obligatory step in the production of key osteoclastogenic signals by in situ osteocytes in fatigue-damaged bone. We found, based on real-time PCR and immunohistochemistry assays, that expression of the apoptosis marker caspase-3 as well osteoclastogenic proteins RANKL and VEGF were increased following fatigue, while expression of the RANKL antagonist OPG decreased. However, when apoptosis was inhibited using QVD, these changes in gene expression were completely blocked. This dependence on apoptosis for neighboring non-apoptotic cells to produce signals that promote tissue remodeling also occurs in response to focal ischemic injury in the brain and heart, indicating that osteoclastic bone remodeling follows a common paradigm for localized tissue repair.

Reductions in serum IGF-1 during aging impair health span.

In lower or simple species, such as worms and flies, disruption of the insulin‐like growth factor (IGF)‐1 and the insulin signaling pathways has been shown to increase lifespan. In rodents, however, growth hormone (GH) regulates IGF‐1 levels in serum and tissues and can modulate lifespan via/or independent of IGF‐1. Rodent models, where the GH/IGF‐1 axis was ablated congenitally, show increased lifespan. However, in contrast to rodents where serum IGF‐1 levels are high throughout life, in humans, serum IGF‐1 peaks during puberty and declines thereafter during aging. Thus, animal models with congenital disruption of the GH/IGF‐1 axis are unable to clearly distinguish between developmental and age‐related effects of GH/IGF‐1 on health. To overcome this caveat, we developed an inducible liver IGF‐1‐deficient (iLID) mouse that allows temporal control of serum IGF‐1. Deletion of liver Igf ‐1 gene at one year of age reduced serum IGF‐1 by 70% and dramatically impaired health span of the iLID mice. Reductions in serum IGF‐1 were coupled with increased GH levels and increased basal STAT5B phosphorylation in livers of iLID mice. These changes were associated with increased liver weight, increased liver inflammation, increased oxidative stress in liver and muscle, and increased incidence of hepatic tumors. Lastly, despite elevations in serum GH, low levels of serum IGF‐1 from 1 year of age compromised skeletal integrity and accelerated bone loss. We conclude that an intact GH/IGF‐1 axis is essential to maintain health span and that elevated GH, even late in life, associates with increased pathology.

Structural and Mechanical Repair of Diffuse Damage in Cortical Bone in vivo

Physiological wear and tear causes bone microdamage at several hierarchical levels, and these have different biological consequences. Bone remodeling is widely held to be the mechanism by which bone microdamage is repaired. However, recent studies showed that unlike typical linear microcracks, small crack damage, the clusters of submicron‐sized matrix cracks also known as diffuse damage (Dif.Dx), does not activate remodeling. Thus, the fate of diffuse damage in vivo is not known. To examine this, we induced selectively Dif.Dx in rat ulnae in vivo by using end‐load ulnar bending creep model. Changes in damage content were assessed by histomorphometry and mechanical testing immediately after loading (ie, acute loaded) or at 14 days after damage induction (ie, survival ulnae). Dif.Dx area was markedly reduced over the 14‐day survival period after loading (p < 0.02). We did not observe any intracortical resorption, and there was no increase in cortical bone area in survival ulnae. The reduction in whole bone stiffness in acute loaded ulnae was restored to baseline levels in survival ulnae (p > 0.6). Microindentation studies showed that Dif.Dx caused a highly localized reduction in elastic modulus in diffuse damage regions of the ulnar cortex. Moduli in these previously damaged bone areas were restored to control values by 14 days after loading. Our current findings indicate that small crack damage in bone can be repaired without bone remodeling, and they suggest that alternative repair mechanisms exist in bone to deal with submicron‐sized matrix cracks. Those mechanisms are currently unknown and further investigations are needed to elucidate the mechanisms by which this direct repair occurs.

Serum IGF-1 is insufficient to restore skeletal size in the total absence of the growth hormone receptor

States of growth hormone (GH) resistance, such those observed in Laron dwarf patients, are characterized by mutations in the GH receptor (GHR), decreased serum and tissue IGF‐1 levels, impaired glucose tolerance, and impaired skeletal acquisition. IGF‐1 replacement therapy in such patients increases growth velocity but does not normalize growth. Herein we combined the GH‐resistant (GHR knockout [GHRKO]) mouse model with mice expressing the hepatic Igf‐1 transgene (HIT) to generate the GHRKO‐HIT mouse model. In GHRKO‐HIT mice, serum IGF‐1 levels were restored via transgenic expression of Igf‐1, allowing us to study how endocrine IGF‐1 affects growth, metabolic homeostasis, and skeletal integrity. We show that in a GH‐resistant state, normalization of serum IGF‐1 improved body adiposity and restored glucose tolerance but was insufficient to support normal skeletal growth, resulting in an osteopenic skeletal phenotype. The inability of serum IGF‐1 to restore skeletal integrity in the total absence of GHR likely resulted from reduced skeletal Igf‐1 gene expression, blunted GH‐mediated effects on the skeleton that are independent of serum or tissue IGF‐1, and poor delivery of IGF‐1 to the tissues. These findings are consistent with clinical data showing that IGF‐I replacement therapy in patients with Laron syndrome does not achieve full skeletal growth.

Characterization of damage mechanisms associated with reference point indentation in human bone

Measurement of bone mineral density (BMD) is the clinical gold standard in cases of compromised skeletal integrity, such as with osteoporosis. While BMD is a useful measurement to index skeletal health, it is also limited since it cannot directly assess any mechanical properties. The ability to directly assess mechanical properties of bone tissue would be clinically important. Reference point indentation (RPI) is a technology that has been designed to try and achieve this goal. While RPI has been shown to detect altered bone tissue properties, the underlying physical mechanism of these measurements has not been characterized. Thus, we designed a study whereby the contribution of (1) test cycle number and (2) test load level to RPI test-induced sub-surface damage was characterized and quantified. Standardized specimens were prepared from cadaveric human tibiae (n=6), such that 12 replicates of each testing condition could be carried out. A custom rig was fabricated to accurately position and map indentation sites. One set of tests was carried out with 1, 5, 10, 15 and 20 cycles (Max Load: 8 N, Freq: 2 Hz), and a second set of tests was carried out with Load levels of 2, 4, 6, 8 or 10 N (Cycle number: 20, Freq: 2 Hz). The RPI parameter Loading Slope (LS) was cycle dependent at 5, 10, 15 and 20 cycles (p<0.05). First Cycle Indentation Distance (ID 1st), Total Indentation Distance (TID), Mean Energy Dissipation (ED), First Cycle Unloading Slope (US 1st), Mean Unloading Slope (US) and LS were significantly different at 6, 8 and 10 N compared to 2 N (p<0.05). From the histomorphometric measurements, damage zone span was significantly different after 5, 10, 15 and 20 cycles compared with 1 cycle while indent profile width and indent profile depth were significantly different at 10, 15 and 20 cycles (p<0.05). With the load varying protocol, each of these parameters differed significantly at each increased load level (4, 6, 8, 10 N) compared with the basal level of 2 N (p<0.05). The damage area parameter in both protocols was significantly different from baseline at the three upper levels tested (i.e. 10, 15, 20 cycles and 6, 8, 10 N, in cycle and load variant protocols, respectively). Specimens were scanned by micro-computed tomography, which showed no material or microstructural differences between samples, and processed for histological analyses and damage quantification. Consistent microdamage patterns were present with evidence of damage via compaction at the indented regions. While damage in the direction of loading was established early, the damage area then increased radially with cycle number. These data help to further understand the physical manifestations of RPI parameters and will help to further facilitate its use as a clinical diagnostic tool.

The Selective Serotonin Re‐Uptake Inhibitor Fluoxetine Directly Inhibits Osteoblast Differentiation and Mineralization During Fracture Healing in Mice

Chronic use of selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression has been linked to osteoporosis. In this study, we investigated the effect of chronic SSRI use on fracture healing in two murine models of bone regeneration. First, we performed a comprehensive analysis of endochondral bone healing in a femur fracture model. C57/BL6 mice treated with fluoxetine, the most commonly prescribed SSRI, developed a normal cartilaginous soft‐callus at 14 days after fracture and demonstrated a significantly smaller and biomechanically weaker bony hard‐callus at 28 days. In order to further dissect the mechanism that resulted in a smaller bony regenerate, we used an intramembranous model of bone healing and revealed that fluoxetine treatment resulted in a significantly smaller bony callus at 7 and 14 days postinjury. In order to test whether the smaller bony regenerate following fluoxetine treatment was caused by an inhibition of osteogenic differentiation and/or mineralization, we employed in vitro experiments, which established that fluoxetine treatment decreases osteogenic differentiation and mineralization and that this effect is serotonin‐independent. Finally, in a translational approach, we tested whether cessation of the medication would result in restoration of the regenerative potential. However, histologic and μCT analysis revealed non‐union formation in these animals with fibrous tissue interposition within the callus. In conclusion, fluoxetine exerts a direct, inhibitory effect on osteoblast differentiation and mineralization, shown in two disparate murine models of bone repair. Discontinuation of the drug did not result in restoration of the healing potential, but rather led to complete arrest of the repair process. Besides the well‐established effect of SSRIs on bone homeostasis, our study provides strong evidence that fluoxetine use negatively impacts fracture healing.

A novel rat model for subchondral microdamage in acute knee injury: a potential mechanism in post-traumatic osteoarthritis

OBJECTIVE Subchondral microdamage may play an important role in post-traumatic osteoarthritis (PTOA) development following anterior cruciate ligament (ACL) rupture. It remains unknown whether this injury mechanism causes subchondral microdamage, or whether its repair occurs by targeted osteoclast-mediated remodeling. If so these events may represent a mechanism by which subchondral bone is involved in PTOA. Our objective was to test the hypothesis that subchondral microdamage occurs, and is co-localized with remodeling, in a novel rat model of ACL rupture. DESIGN We developed a novel non-invasive rat animal model for ACL rupture and subchondral microdamage generation. By inducing ACL rupture noninvasively rather than surgically, this more closely mimics the clinical injury. MicroCT, MRI and histological methods were used to measure microstructural changes, ligament damage, and cellular/matrix degeneration, respectively. RESULTS We reproducibly generated ACL rupture without damage to other soft joint tissues. Immediately after injury, increased microdamage was found in the postero-medial aspect of the tibia. Microstructural parameters showed increased resorption at 2 weeks, which returned to baseline. Dynamic histomorphometry showed increased calcein label uptake in the same region at 4 and 8 weeks. Chondrocyte death and protease activity in cartilage was also noted, however whether this was directly linked to subchondral changes is not yet known. Similarly, cartilage scoring showed degradation at 4 and 8 weeks post-injury. CONCLUSIONS This study shows that our novel model can be used to study subchondral microdamage after ACL-rupture, and its association with localized remodeling. Cartilage degeneration, on a similar time-scale to other models, is also a feature of this system.

The roles of osteocyte signaling in bone.

Osteocytes are the cells that reside within the bone matrix and make up 90% to 95% of the cellular component of the tissue. Until recently, they were thought to serve as little more than “space holders” in the tissue. However, significant advances have demonstrated that osteocytes are of the utmost importance in regulating the dynamic nature of bone; indeed, it may be that this is their major function. Specifically, osteocyte signaling in skeletal metabolism contributes to (1) regulation of local mineralization, (2) regulation of systemic mineralization, (3) bone formation by osteoblasts, and (4) bone resorption by osteoclasts.