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.
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 .
- 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%)
- 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].
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 , 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.  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.
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.
This HubLE Method is about all the morphometric parameters that microCT can measure in bone. What do they mean? Which ones are important for my genetic / disease model?
- uCT scanner, with powerful PC: min 32Gb RAM, preferably Nvidia GPU.
- Reconstructed uCT bone datasets
- Software for 3D morphometric analysis of bone including flexible VOI selection and thresholding / segmentation (making black and white images) and 3D analysis equipment
- For characterisation of trabecular and cortical bone, in osteopenia studies or general bone phenotype studies, the VOI is standardised relative to the growth plate2.
- For study of subchondral bone at the knee articulation between femur and tibia in arthritis, the appearance of a connecting bridge in cross-section between the two condyles can be used as a reference point.
- For study of bone disruption (osteolysis and pathological formation) by bone tumour metastases or myeloma, a segment of the metaphysis should be selected relative to either the growth plate or the end of the bone (junction of condyles). Note that severe osteolysis can complicate VOI selection since large parts of bone structures can be missing.
- For study of a fracture callus, identify the fracture midline and choose a fixed number of cross-section slices on either side of this point, such as 100-200 slices on both sides.
- For cartilage, for instance if a rodent knee is stained by a contrast agent such as phospho-tungstic acid, regions of the medial condyle articular cartilage can be selected where the condyles are separated, not touching each other, for easier VOI selection.
- Drilled bone cores need only a cylindrical VOI in the middle of the specimen to avoid damaged tissue.
- Use 3D software tools to delineate the VOI, either by manual drawing or automatic delineation, carefully referencing the VOI to anatomical landmarks3
Parameter name; ASBMR nomenclature4 symbol; Unit; What it is, how to use it:
Bone volume; BV; mm3; The absolute quantity of bone by volume, within your VOI.
(Remember – a consistent VOI is important!). The 2D equivalent is cross-sectional bone area, BA.
- Percent bone volume; BV/TV; %; If your VOI is fully contained within bone, such as a region of medullary marrow space containing trabecular bone, then BV/TV is the key parameter of the spatial density or occupancy of that VOI. Note however that if a VOI extends beyond bone arbitrarily into surrounding space, such as a VOI drawn around the outside of cortical bone or a fracture callus, then BV/TV is not relevant. The 2D equivalent is % bone area to total area, BA/TA.
- Bone surface to volume ratio; BS/BV. mm-1; A useful general indicator of structural complexity and inversely related to bone or trabecular thickness. Can be in 2D (perimeter / area) or 3D (sfc. / vol.).
- Trabecular (Cortical) thickness; Tb.Th (Ct.Th); mm; 3D thickness measured by virtual sphere-fitting.
- Trabecular separation (pore diameter); Tb.Sp (Po.Dm); mm; Diameter of spaces measured as Tb.Th.
- Trabecular number; Tb.N; mm-1; The spatial density of structures within a VOI such as trabeculae. It is computed as the number of times per mm a random line through a VOI crosses a trabecula. Tb.N can be calculated from (BV/TV)/Th but a 3D calculation as 1/(Tb.Th+Tb.Sp) is more accurate.
- Trabecular pattern factor; Tb.Pf; mm-1; Alternatively called surface convexity index (SCv.I). Convex curvature moves the index in a positive direction, while concave curvature makes the index negative (flat is zero). It is an inverse indicator of connectivity since multiply connected structures – such as healthy trabecular bone – have a lot of concave curvature at their nodes and thus low or negative values. Tb.Pf and SMI (next) are measured by the same differential method and are useful measures of “texture” of fracture callus and pathological ectopic bone formed in arthritis and tumour models.
- Structure model index; SMI; None; SMI like Tb.Pf measures surface curvature. In ideal shapes a flat plate, a cylinder and a sphere have SMI of 0, 3 and 4 respectively. SMI has been criticised (5) for expectation of these ideal shape model values ignoring concave curvature of node connections, and for its % volume bias; however it remains a useful parameter of architecture if correctly interpreted.
- Fractal Dimension; FD; None; This parameter looks for repetition of similar structures on different spatial scales – like a fern leaf. A useful general index of complexity in bone disruption models.
- Euler Connectivity (density); Conn (Conn.D.); None (mm-3); Euler topological connectivity counts the number of loops or alternative connections. It can show loss of connections in osteoporotic bone.
- Degree of Anisotropy; DA; None; Are trabecular structures aligned in one direction – e.g. of mechanical loading? Then DA is high. If not, DA is low. DA increases as fracture callus remodels to cortical bone.
- Moment of Inertia parameters; MMI; mm4; MMI parameters are defined around an axis. This can be the X or Y axis of a cross-section plane, or the Z axis. It helps if your software calculates the maximum and minimum axes of inertia (strongest and weakest direction). Things break in their weakest axis. There are many MMI indices such as x, y, polar, principal min and max.
Salmon PL (2020) Micro-computed Tomography (micro-CT) in Medicine and Engineering; Orhan K ed, Springer Nature Switzerland, pp. 49-75.
van ‘t Hof RJ, Dall’Ara E. Analysis of Bone Architecture in Rodents Using Micro-Computed Tomography. Methods Mol Biol. 2019;1914:507-531.
Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, & Muller R. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone and Miner Res, 2010;25:1468–1486.
Dempster DW, Compston JE, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR, Parfitt AM (2013) Standardized nomenclature, symbols, and units for bone histomorphometry: a 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee. Journal of Bone and Mineral Research 28(1): 2-17.
- Salmon PL, Ohlsson C, Shefelbine SJ, Doube M (2015) Structure Model Index Does Not Measure Rods and Plates in Trabecular Bone. Front Endocrinol (Lausanne). 2015; 6: 162.