Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
Osteoporosis is a bone disease that causes decreased bone quality and strength, resulting in increased fracture risk. Currently, anabolic, bone-forming therapies are limited, highlighting the need for novel therapeutic options. Mechanical loading of the skeleton produces anabolic tissue responses and increases bone mass clinically and in preclinical models. This anabolic tissue response is driven by a cascade of osteogenic signaling pathways, few of which have been identified, suggesting that more remain to be discovered. Transcriptomic profiles provide insight into biological pathways activated by mechanical loading. Tibial compression produces varying deformation magnitudes along the axial direction of the cortex, inducing the highest strains at the mid-diaphysis and the lowest at the metaphyseal shell. Anabolic pathways in the cortical bone that are differentially activated based on strain magnitude may identify promising novel therapeutic targets and have not been explored in previous transcriptomic studies.
In our study published in JBMR (2022), we sought to elucidate the role of mechanical strain magnitude on the transcriptional response of cortical bone during loading. The left limb of female mice was loaded in compression, and the right limb served as the contralateral control. Gene expression was evaluated at early time points following a single bout of loading (1 h, 3 h, and 24 h) or at 1 wk following daily bouts of loading. Taking advantage of the natural gradient of strain induced by cyclic tibial compression, we isolated RNA from three strain regions in the cortex: metaphyseal cortical shell (low strain), proximal diaphysis (medium strain), and mid-diaphysis (high strain). Differential gene expression was analyzed between loaded and control limbs, correlated with enrichment of biological processes, and validated with in situ hybridization. We found that transcriptomic responses correlated with tissue strain magnitude; at each time point, the mid-diaphysis (highest strain) had the most differentially-expressed genes and the metaphyseal cortical shell (lowest strain) had the least. Similarly, biological processes regulating bone formation and turnover increased earlier and to the greatest extent at the mid-diaphysis.
Higher strain induced greater levels of osteocyte-associated genes (Sost, Mepe), whereas expression was lower in osteoclast-related genes (Ctsk, Acp5). The differentially-expressed genes and biological processes were unique across all three tissue segments. Finally, the distinct transcriptomic responses recorded at each time point following loading highlight the complex cascade of osteogenic signaling induced by loading.
In summary, cortical bone responded to mechanical loading as a function of strain magnitude across early and late time points. In cortical bone, higher strain magnitudes elicited larger, earlier anabolic responses in cortical bone whereas low strain magnitude was correlated with a diminished response. This research highlights the importance of spatial evaluation of transcriptional profiles. Future work using bulk RNA sequencing should distinguish the cortical segment by axial location or mechanical strain experienced. Finally, this work enhanced our understanding of the role of mechanical strain in the transcriptomic response of cortical bone to loading, thereby improving the ability to create effective targeted therapeutics.
This work was conducted by Carolyn Chlebek, Jacob Moore, F. Patrick Ross, and Marjolein van der Meulen. Funding for this research was provided by NSF Grant #1636012, NSF GRFP (DGE-1650441), and GAANN P200A150273 J. The authors would like to thank Dr. Adrian McNairn and Dr. John Schimenti for their experimental assistance and for providing essential training that enabled this research. For assistance with analysis, the authors acknowledge the Cornell University Bioinformatics Facility and specifically Dr. Qi Sun. Danielle Jorgenson also assisted in data analysis and sorting. The authors also acknowledge the Cornell CARE staff. Transcriptomic data presented in this study are available in Gene Expression Omnibus, Accession GSE210827.
Osteoporosis, which predominantly occurs in females, affects 200 million women worldwide and is characterised by bone loss and microarchitectural degradation, which leads to fractures, pain and immobility. Conventional treatments for osteoporosis target bone loss by inhibiting osteoclast activity, but these only prevent osteoporotic fractures in 50% of sufferers. An advanced understanding of the underlying mechanisms and treatments for osteoporosis is required to avert the projected trend, whereby the worldwide economic burden of treatment will reach $132 billion by 2050. Osteoblasts and osteocytes are known to regulate the differentiation, function and survival of osteoclasts, through expression of paracrine factors, which can be altered following mechanical loading and are affected by the presence of estrogen. Studies have recently revealed changes in osteoblast and osteocyte mechanobiology during estrogen deficiency, but, how these changes effect osteoclastogenesis is not fully understood. The first study of this thesis sought to assess changes in osteoblast-induced osteoclastogenesis under combined estrogen deficiency and mechanical loading, using a combination of conditioned media and co-culture experiments. MC3T3-E1 cells were cultured with pre-menopausal levels of estrogen which was then withdrawn from the media and the osteoblast like-cells were exposed to oscillatory fluid flow. These studies found that conditioned media from estrogen-treated MC3T3-E1 cells inhibited the differentiation of RAW264.7 cells and inhibited podosome belt formation. Estrogen deficient osteoblasts down-regulated osteoprotegerin (OPG) expression in response to fluid flow when compared to estrogen-treated osteoblasts, leading to a significant increase in the ratio of RANKL/OPG gene expression. Subsequently, an increase in osteoclast number and an up-regulation in nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and Cathepsin K expression was observed in RAW264.7 cells. In addition, estrogen deficient osteoblasts co-cultured with RAW264.7 cells resulted in an increase in osteoclastogenesis and matrix degradation compared to co-cultures with estrogen-treated osteoblasts. Additionally, it was found that Rho-ROCK inhibition in osteoblasts, exacerbated the increase in osteoblast-induced osteoclastogenesis that arose under estrogen deficiency. It is proposed that the reduction in OPG production by mechanically stimulated osteoblasts during estrogen deficiency may leave osteoclast differentiation and matrix degradation activity unchecked and, thereby, play an important role in bone loss during osteoporosis. The second study sought to establish whether mechanically stimulated osteocytes induce osteoclastogenesis and bone resorption during estrogen deficiency. This study built upon the post-menopausal model and co-culture methods established in the first study. The findings revealed that under postmenopausal conditions RANKL/OPG gene expression is increased in mechanically stimulated osteocytes (OCY454 cells) compared to estrogen-treated osteocytes and a significant increase in osteocyte-induced osteoclast formation (by bone marrow macrophage cells) occurs leading to increased resorption. Estrogen deficient osteocytes also up-regulated sclerostin expression following mechanical loading. Interestingly, Wnt antagonists WIF1 and FRZB were down-regulated in estrogen deficient osteocytes following loading. A second aim of this study was to investigate whether a neutralising antibody against sclerostin (Scl-Ab) could revert osteocyte-mediated osteoclastogenesis and resorption by attenuating RANKL/OPG gene expression. It was demonstrated that administration of the Scl-Ab reduced pro-osteoclastogenic signalling (RANKL/OPG) between osteocytes and osteoclasts, which led to reduced resorption. Osteocytes are housed within lacunae and their dendrites extend through canaliculae to enable the formation of gap junctions with neighbouring osteocytes and osteoblasts. The lacunar-canalicular network and vascular pores facilitate movement of interstitial fluid, which confers mechanical stimulation on osteocytes and facilitates transport of nutrients and signalling molecules to and from the osteocyte. There is some evidence that this microporous network is altered during estrogen deficiency, which might alter fluid flow through the network and the ensuing responses of osteoblasts and osteocytes. While previous studies have investigated early-stage estrogen deficiency, the long term effects of estrogen deficiency and the time-sequence of changes in cortical bone microporosity are not known. For this reason, the final study of this thesis sought to determine the temporal changes that occur in the cortical vasculature and the lacunar-canalicular network during short and longer term estrogen deficiency. This study quantified microporosity and lacunar occupancy in an ovariectomised rat model of osteoporosis by micro-CT analysis, backscatter electron imaging and histological analysis. It was shown that initial increases in canalicular diameter and vascular porosity arose during short term estrogen deficiency (week 4 OVX) compared to aged-matched controls, but that these were reduced along with a decrease in lacunar diameter and lacunar occupancy in long term estrogen deficiency (week 14 OVX). These changes could be explained by perilacunar remodelling, micropetrosis or a mechanobiological adaptive response, such as increases in bone mass and bone mineralisation that may occur to alter the mechanical environment in an attempt to restore tissue homeostasis. This study also demonstrated that administration of Scl-Ab to ovariectomised rats resulted in a significant decrease in osteoclast number and prevented the occurrence of empty lacunae in long term estrogen deficiency (week 14 OVX) compared to untreated week 14 OVX animals. Together, the studies in this thesis found that the inhibitory effects mechanically loading has on pro-osteoclastogenic signalling in osteocytes and osteoblasts is attenuated during estrogen deficiency. Using cell culture and mechanobiological techniques, the changes in paracrine factor expression in estrogen deficient osteoblasts and osteocytes and their implications in terms of osteoclastogenesis and resorption were elucidated. Additionally, the temporal changes in cortical microporosity that occur were established. Collectively, the research presented in this thesis provides an important, but previously unrecognised, insight into the mechanobiological changes that occur during estrogen deficiency leading to bone loss. The information gained from this work may inform future mechanobiological treatments for osteoporosis.
Full text of D. Allison’s thesis is available here.
The pericellular matrix (PCM) of cartilage is a structurally distinctive microdomain surrounding each chondrocyte, and is pivotal to cell homeostasis and cell-matrix interactions in healthy tissue. This study queried if the PCM is the initiation point for disease or a casualty of more widespread matrix degen- eration. To address this question, we queried the mechanical properties of the PCM and chondrocyte mechanoresponsivity with the development of post-traumatic osteoarthritis (PTOA). To do so, we inte- grated Kawamoto’s film-assisted cryo-sectioning with immunofluorescence-guided AFM nanomechanical mapping, and quantified the microscale modulus of murine cartilage PCM and further-removed extracel- lular matrix. Using the destabilization of the medial meniscus (DMM) murine model of PTOA, we show that decreases in PCM micromechanics are apparent as early as 3 days after injury, and that this pre- cedes changes in the bulk ECM properties and overt indications of cartilage damage. We also show that, as a consequence of altered PCM properties, calcium mobilization by chondrocytes in response to me- chanical challenge (hypo-osmotic stress) is significantly disrupted. These aberrant changes in chondro- cyte micromechanobiology as a consequence of DMM could be partially blocked by early inhibition of PCM remodeling. Collectively, these results suggest that changes in PCM micromechanobiology are lead- ing indicators of the initiation of PTOA, and that disease originates in the cartilage PCM. This insight will direct the development of early detection methods, as well as small molecule-based therapies that can stop early aberrant remodeling in this critical cartilage microdomain to slow or reverse disease progression.
Full text of Dr D. Chery’s article is available from HERE