38 Biomechanics of Bone Cells
Mechanosensitive bone cells translate mechanical stimuli into a biological response. The bone cell response to mechanical stimuli varies from one individual to another depending on age, sex, as well as physiological and pathological conditions. 1 In the absence of mechanical stimuli, such as during disuse, stasis of interstitial fluid in bone occurs leading to a lack of fluid shear stress on the osteocytes. 2 Osteocytes produce pro-osteoclastogenic signals in the absence of mechanical loading, leading to a stimulation of bone resorption. 3 In the presence of mechanical stimuli, osteocytes produce factors that inhibit osteoclastogenesis and/or decrease the production of osteoclast-stimulating signals. 4 The most well-known soluble factors affecting osteoclastogenesis are receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), which stimulate osteoclast formation, and osteoprotegerin (OPG), which inhibits osteoclast formation. Under disuse conditions, osteocytes produce RANKL, M-CSF, and OPG. In response to mechanical stimulation, osteocytes produce factors (e.g., matrix extracellular phosphoglycoprotein) that decrease RANK, and increase OPG production 5 (Fig. 38.1).
To study the communication between mechanically stimulated osteocytes and osteoclast precursors in vitro, one requires a source of osteocyte-like cells, a method for mechanical stimulation, and a source of osteoclast precursors. Mechanical stimuli can be applied to osteocytes in vitro through various methods, such as pulsating fluid flow (PFF). The protocol for performing PFF is described in Chapter 37. Osteocyte-like cells of human origin can be obtained by culturing human primary bone cells as outgrowth from long bones. Because osteocytes are terminally differentiated cells of the osteoblast lineage, mechanosensitive human osteoblast cell lines can be used as a model for osteocytes. In studies where it is appropriate to use cells of animal origin, the mouse osteocyte cell line MLO-Y4 can be used or osteocytes can be isolated from the calvariae of chickens. Basic principles of culturing bone cells are described in Chapter 35. Bone marrow and peripheral blood mononuclear cells (PBMC) are an excellent source of osteoclast precursors. PBMCs can be easily isolated from human peripheral blood, and when cultured with RANKL and M-CSF, these cells fuse and form multinucleated osteoclasts. 6, 7 The formation of osteoclasts in vitro can be quantified relatively easily as osteoclasts are multinucleated and produce high levels of the enzyme tartrate-resistant acid phosphatase (TRAcP), which can be visualized by a TRAcP staining.7, 8 In addition, PBMCs cultured on a bone or dentin slice become active, bone-resorbing osteoclasts and form a resorption pit on the slice. These resorption pits can be visualized by a Coomassie Brilliant Blue (CBB) staining after removal of the cells 9 (Fig. 38.2).
Culture medium taken from PFF-stimulated or statically cultured osteocytes (conditioned medium) contains growth factors produced by the osteocytes. Control medium (medium that has not been in contact with osteocytes) lacks osteocyte-growth factors. Compared to control medium, culturing osteoclast precursors with static conditioned medium enhances osteoclast formation, whereas culturing osteoclast precursors with conditioned medium of PFF-stimulated osteocytes reduces osteoclast formation.5
In the first half of this chapter, we focus on in vitro methods that can be used to analyze the effect of conditioned medium of osteocytes on osteoclast formation and osteoclast activity. In the second half of this the chapter, we focus on in vitro methods for analyzing the effect of conditioned medium on proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs). Under physiological conditions, osteocytes produce signaling factors that enhance osteogenic differentiation of stem cells (e.g., bone morphogenic proteins and Wnts) in response to mechanical loading 10 (Fig. 38.3). MSCs are the progenitors of bone-forming cells. They are often derived from bone marrow or adipose tissue for in vitro experiments.
In this chapter, we describe a protocol for the culture of human bone marrow–derived MSCs with conditioned medium of osteocytes or control medium, and the subsequent analysis of proliferation and osteogenic differentiation of the MSCs. Cell proliferation is estimated by measuring the cell number via quantification of deoxyribonucleic acid (DNA) content. Osteogenic differentiation of MSCs can be determined by quantifying gene expression of osteogenic markers via polymerase chain reaction (see Chapter 39). Mature, active osteoblasts deposit mineralizing matrix in culture, which can be visualized by alizarin red or von Kossa-staining. Osteocalcin and serum procollagen type 1 amino-terminal propeptide are released in the culture medium and can be analyzed by enzyme-linked immunosorbent assay (Fig. 38.4).
38.1 Materials and Reagents
All reagents, materials, and culture medium used for cell purification and cell culture should be sterile.
38.1.1 Osteoclastogenesis and Osteoclast Activity Assays
Osteoclast Precursor (Peripheral Blood Mononuclear Cell) Isolation
Buffy coat or venipuncture blood with anticoagulant (e.g., ethylenediaminetetraacetic acid or heparin).
1% phosphate buffered saline (PBS)-citrate: Mix 10 mL citrate stock in 1,000 mL PBS at room temperature (RT), and put the bottle on ice until use.
Citrate stock: Dissolve 456 g of sodiumcitrate-dihydrate (molecular weight = 294) and 21.4 g of citratemonohydrate (molecular weight = 192.12) in up to 1 L of Milli-Q water (Millipore Corporation, Billerica, MA) and autoclave.
Lymphoprep (Ficoll).
50 mL tubes.
Pasteur pipettes.
Cell culture flasks (T75) and 96-well plates.
Peripheral Blood Mononuclear Cell Culture
Dulbecco′s Modified Eagle′s Medium (DMEM)
Fetal clone serum (FCS) (see Note 38.1 (p. 307))
Penicillin-streptomycin-fungizone (PSF; Sigma #A-5955, St. Louis, MO)
M-CSF (R&D Systems #216-MC, Minneapolis, MN)
RANK-L (Peprotech #310–01, Rocky Hill, NJ)
Conditioned medium: Collect the conditioned medium after 60 minutes of PFF/static culture of primary human bone cells and store the medium at −20°C (see Note 38.2 (p. 307))
Control medium: DMEM + 10% FCS + 1% PSF + M-CSF (50 ng/mL) + RANK-L (80 ng/mL); make fresh control medium each time
96-well culture plates (Greiner # 655180, Monroe, NC) (see Note 38.3 (p. 307))
Tartrate-resistant Acid Phosphatase Staining
Leukocyte acid phosphatase TRAcP kit (Sigma #387A-1 kt)
PBS-buffered 4% formaldehyde
Milli-Q water
Potassium sodium tartrate solution (1 mol/L); dissolve 2.8 g in 10 mL Milli-Q water
4′, 6-diamidino-2-phenylindole (DAPI), stock = 100 mg/mL in PBS, dilute 100 × to make a working solution (see Note 38.4 (p. 307))
Bone Resorption Visualization
Bovine cortical bone slices (thickness 0.5 mm).
PBS.
70% ethanol.
Medium: DMEM + 10% FCS + 1% PSF.
Forceps.
10% ammonium hydroxide (NH4OH) solution.
Approximately 10% water saturated alum [KAl(SO4)2*12H2O]; filter before use.
CBB (PhastGel Blue R, Pharmacia, Uppsala, Sweden). Dissolve 1 tablet in 80 mL water for 5 minutes and add 120 mL methanol. The solution is stable for a couple of months in the refrigerator. Dilute 1:1 in 20% acetic acid directly before use. Filter this solution (Wattman) before use.
38.1.2 Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells
Mesenchymal Stem Cell Culture
Human MSCs, bone marrow (Lonza Cologne GmbH, Long Branch, NJ).
DMEM.
FCS.
PSF (Sigma #A-5955).
20 mM β-glycerophosphate (Sigma); dissolve 14.79 g/50 mL PBS (1 M), heat at 50°C for 10 minutes to dissolve. Filter and divide in 1 mL aliquots, and store at −20 °C. Dissolve in 50 mL medium to make a final concentration of 20 mM.
100 µM ascorbic acid (Sigma); dissolve at 5 mg/mL PBS, filter, and add 1 mL to 50 mL medium to make a final concentration of 100 µM (make fresh solution before each use).
48-well culture plates.
Deoxyribonucleic Acid Quantification
PBS
Milli-Q water
CyQUANT NF Cell Proliferation Assay Kit (Invitrogen, Carlsbad, CA)
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