Kris Kaleta
Molecular Identification of Spatially Distinct Anabolic Responses to Mechanical Loading in Murine Cortical Bone
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.
VEGFA’s critical role in blood vessel formation during bone repair is cell type and injury specific
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Formation of new blood vessels is necessary for proper bone fracture healing. In support of this, fracture patients with pre-existing conditions associated with vascular disease and injury, such as smoking, advanced age, diabetes or other large-scale soft tissue trauma have lower successful fracture healing rates. Vascular endothelial growth factor A, abbreviated as VEGFA, is essential in coordinating the proliferation and recruitment of existing blood vessels across a range of bodily injury sites. In bone, blocking VEGFA with certain pharmacological agents, has been shown to inhibit bone fracture healing. However, since these pharmacological approaches are systemic and nonspecific, the cells in bone that make VEGFA and are necessary for blood vessel formation during fracture healing, remain poorly defined. This work is important to understanding why certain fractures won’t heal and would inform future treatment strategies.
Recent work in the role of blood vessels in bone came from Henry Kronenberg and Crista Maes at Harvard. These authors demonstrated that VEGFA expressing Osterix (Osx) lineage cells, a marker of early osteoprogenitors which give rise to bone forming cells called osteoblasts, secrete VEGFA and invade the fracture callus with blood vessels. Furthermore, the team of Bjorn Olsen at Harvard, demonstrated via genetic deletion of VEGFA from Osx lineage cells that blood vessel formation during cortical defect healing was impaired. However, this localized bone injury doesn’t recapitulate all aspects of fracture repair, including periosteal activation in the outer lining of the bone. Building upon these important works, our lab group at WUSTL decided to test the role of VEGFA from osteoprogenitors (Osx lineage) and their more mature cells osteoblasts and osteocytes, marked by Dentin Matrix Protein 1 (DMP1), during clinical fracture repair models such as stress fractures and full fracture.
In our study, published in JBMR in 2019, we performed genetic inducible deletion of VEGFA in different cell types. We deleted VEGFA from all cells, or from bone-forming osteoblast cells at 2 stages of maturation; osteoprogenitors and mature osteoblasts/osteocytes. We then performed either a full semi-stabilized fracture or stress fracture. Cortical defect healing was also tested to compare to the results of the Olsen team. Our study revealed that loss of VEGFA in all cells blocked bone and blood vessel formation regardless of the injury model, reaffirming previous pharmacological results. More importantly, we showed that VEGFA from osteoprogenitor cells (Osx lineage) but not more mature osteoblasts/osteocytes (DMP1 lineage) was necessary for maximal bone formation and blood vessel formation in stress fracture and full fracture models. Surprisingly, VEGFA from osteoprogenitor cells at the time of injury was not important for cortical defect repair. These results together, highlight that the VEGFA cell source necessary for bone healing depends on the type of bone injury. Furthermore, our study is the first to indicate that VEGFA from different osteoblast maturation stages play nonredundant roles during clinical models of fracture healing. Our results may also help explain why recombinant BMPs, which stimulate VEGFA production in osteoprogenitors, are promising treatments to induce bone formation during fracture. Overall, these findings highlight that osteoprogenitors are critically sources of VEGFA during fracture healing. Furthermore, these same cells should be clinical targets for improving bone blood vessel formation to promote fracture repair in patients with poor healing outcomes.
These findings are described in more detail in the article entitled “VEGFA From Early Osteoblast Lineage Cells (Osterix+) Is Required in Mice for Fracture Healing” published in JBMR. The work was conducted by Evan Buettmann, Jennifer McKenzie, Nicole Migotsky, David Sykes, Pei Hu, Susumu Yoneda, Matthew Silva. This work was funded by NIAMS (R01 AR050211 and P30 AR057235). The authors would like to thank the Washington University in St. Louis Musculoskeletal Research Center (MRC) Cores and staff for histological and micro-CT imaging assistance. Histological images were taken with the Nanozoomer at Alafi Neuroimaging Core (S10 RR027552). VEGFAfl/fl mice from Genentech (Roche Holding AG) were kindly provided by the lab of Dr. Bjorn Olsen (Harvard). Inducible Osx Cre-ERT were generated and provided by the lab of Dr. Henry Kronenberg (Harvard). Inducible DMP1-Cre-ERT2 generated by the lab of Dr. Paola Divieti Pajevic (Boston University) were kindly provided by the lab of Dr. Alexander Robling (Indiana University Medical School).
Mendelian randomization: evaluation of causality between risk factors and outcomes
Graphical Abstract
Abstract
Mendelian randomization (MR) is a powerful approach that evaluates the causal association between a risk factor and an outcome. It makes use of the random allocation of genetic variants to mimic randomizers in randomized controlled trials (RCTs), providing quality evidence that is less susceptible to unmeasured confounding and reverse causality, when compared to conventional observational studies. Currently, MR has been applied in osteoporosis-related research to begin to unravel the causal risk factors that predispose to low bone mineral density (BMD) and increased susceptibility of fracture. Some MR studies made use of serum level measurement as a surrogate to mimic the role of supplementation, such as vitamin D and calcium, and evaluate the effects of the supplements in bone metabolism. From clinical perspective, MR studies enable identification of diagnostic markers and therapeutic targets. They provide evidence on the efficacy and adverse effects of drugs, contributing to discovery and repurposing of drugs.
Article
Culture of the IDG-SW3 osteocyte cell line
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Introduction
The IDG-SW3 osteoblast-to-late-osteocyte cell line is derived from a temperature-sensitive DMP1-GFP transgenic mouse. IDG-SW3s cultured with interferon g (IFNγ) at 33oC will proliferate, whilst culture in mineralising conditions without IFNγ at 37oC enhances differentiation. This HubLE Method describes the protocol for culturing this cell line [1].
Materials
- Rat-tail type 1 collagen
- Recombinant mouse interferon g (IFNγ)
- Phosphate buffered saline (PBS)
- Proliferation medium Alpha Modified Essential Medium (ProlifαMEM) [Tip No. 1]
- Osteocyte-differentiation αMEM (OcyMEM) [Tip No. 2]
- Trypsin-EDTA (0.25%)
- Glutaraldehyde (2.5%)
- Alizarin Red (1%)
Methods [Update]
All procedures should be performed under sterile conditions.
- Coat all required tissue culture plastics for 1 hour at room temperature with 0.15mg/ml of rat-tail type 1 collagen in 0.02M acetic acid.
- Remove the collagen solution and either wash with PBS for immediate use, or air dry plates prior to storage [Tip No. 3].
- Thaw a vial of IDG-SW3 cells into 5ml ProlifαMEM and spin at 1,500 rpm for 5 minutes.
- Remove the supernatant, re-suspend the pellet in ProlifαMEM and seed into a collagen-coated 75cm2 flask containing ProlifαMEM. Incubate at 33oC with 5% CO2.
- Once ≥80% confluent (2-3 days post-seeding), remove medium, wash with PBS and incubate with 0.25% Trypsin for 5-10 minutes to detach cells.
- Spin at 1,500g for 5 minutes and re-suspend in ProlifαMEM (1ml per flask).
- Seed cells in collagen-coated tissue culture trays or flasks (for further expansion and use of cells at next passage) in ProlifαMEM [Tip No. 4].
- Incubate at 33oC with 5% CO2 until confluent.
- At this stage, remove the ProlifαMEM medium, carefully wash the cell monolayers with PBS and add OcyMEM. Incubate at 37oC with 5% CO2.
- Culture for up to 30-35 days with half medium changes of OcyMEM every 2-3 days. Mineralisation is usually evident from ~10-14 days.
- SFix with 2.5% glutaraldehyde for 5 minutes before staining with 1% alizarin red to visualise mineralised nodules [Tip No. 5].
Tips [Update]
ProlifαMEM: Add 10% heat-inactivated foetal calf serum (FCS), AB/AM (100U/ml penicillin, 100mg/ml streptomycin, 0.25mg/ml amphotericin) and L-glutamine (200mM). Aliquot the stock media and add IFNγ (2500U/ µl). Incubate the medium at 33oC prior to use and limit exposure to heat due to IFNγ degradation.
OcyMEM: Add 10% heat-inactivated foetal calf serum (FCS), AB/AM (100U/ml penicillin, 100mg/ml streptomycin, 0.25mg/ml amphotericin) and L-glutamine (200mM). Add 50µg/ml ascorbate and 2-4mM β-glycerophosphate (the original paper uses 4mM β-GP [1], however, IDG-SW3 cells differentiate and mineralise sufficiently in 2mM). Always make fresh on the day of use.
Collagen-coated tissue culture plates/ flasks: Ensure all plates are coated under sterile conditions in a tissue culture hood. Use a cold pipette (stored in the freezer until use) to stop the collagen sticking to the plastic. The 0.15mg/ml collagen solution can be re-used 5-6 times; coating for ~1 hour each time. Coated plastics wrapped in parafilm can be stored at 4oC for up to 6 months until use.
Seeding density: IDG-SW3 cells will mineralise in 12 and 6-well plates but due to the long culture duration some monolayer peeling should be expected. Woo et al. [1] recommend seeding IDG-SW3 cells at 4×104 cells/cm2, although the lower densities of 104 (12-well) and 105 (6-well) will also support osteocyte proliferation, mineralisation and differentiation. To expand IDG-SW3 cells for the subsequent passage seed at 5×105 cells/ 75cm2 flask.
Alizarin Red staining: Mineralised bone nodules can be stained with alizarin red (Fig.1C). It is also possible to obtain good quality images on unstained cell layers (Fig 1A-1B). DMP1-GFP expression can be visually monitored throughout the differentiation process (Fig.1D). Evaluation of E11, DMP1 and sclerostin gene/ protein expression is also advisable to confirm osteocyte differentiation.
References [Update]
Woo SM, Rosser J, Dusevich V, Kalajzic I, Bonewald LF (2011). Cell line IDG-SW3 replicates osteoblast-to-late osteocyte differentiation in vitro and accelerates bone formation in vivo. J Bone Mineral Res 26:2634-2646.
Capitalising on the use of bone-modulating drugs in the treatment of breast cancer metastasis
Infographic
Abstract
Most breast cancer patients have no evidence of metastasis at the time of original diagnosis, yet ~30% of patients experience recurrent disease. Over 90% of cancer deaths occur due to metastasis, and bone is the most common site of breast cancer metastasis. In some clinical trials, adjuvant zoledronic acid (ZA), a bone-targeting agent, increased disease-free survival but responses were nevertheless limited. The reasons for limited responses are unknown and the mechanisms underlying ZA’s protective effect are unclear. Here, using preclinical breast cancer metastasis models, we establish that bone marrow hematopoietic cells harbor tumor suppressive activity in response to ZA. Specifically, ZA renders myeloid/osteoclast progenitor cells (M/OCPs) tumor-suppressive by altering their gene expression profile and lineage potential. Granulocyte-colony stimulating factor (G-CSF) counteracts ZA’s beneficial effects by directing M/OCP differentiation toward osteoclasts, which ablates metastasis suppression. Women enrolled in a clinical trial who had plasma G-CSF levels >23 pg/mL at randomization experienced a significant reduction in disease-free survival with adjuvant ZA. Our study lays a foundation for understanding breast cancer patient responses to ZA and suggests that finding ways to capitalize on M/OCP function and differentiation potential would constitute novel therapeutic approaches to prevent or limit metastatic disease in the bone.
Thesis
The full text of Dr Jessalyn Ubellacker’s thesis is unavailable.
Imaging of skeletal muscle cells and extracellular matrix
Confocal fluorescent microscope showing tissue cross section of healthy porcine skeletal muscle stained using phalloidin (red muscle cells) and wheat germ agglutinin (green, extracellular matrix).
Image acquired on a Nikon Eclipse E800 microscope with Nikon COOLPIX P600