Name: a novel in vivo gene transfer technique and in vitro cell based assays for the study of bone loss in musculoskeletal disorders
Uploaded: 2014-06-08T23:30:00.000+00:00
Duration: 11 min 47 s
Description: Watch this Scientific Journal Video about A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders at JoVE.com

Research was partly supported by NIH research grants R01 AR062173 and SHC 250862 to IEA. ES is the recipient of NIH T32 CTSC predoctoral fellowship.

1. Hydrodynamic Delivery of sRANKL MC DNA
<ol>
	<li>Hydrodynamic Delivery via Mouse Tail Vein
	<ol>
		<li>Weigh the mouse before the tail vein injection. Dilute sRANKL or green fluorescent protein (GFP) MC in Ringer's solution (pre-warm at 37 &#176;C) in a total volume of ~10% of the mouse's body weight.</li>
		<li>Warm up the mouse in a cage for 10 min prior to injection in order to dilate blood vessels and make lateral veins (LVs) visible. Monitor the mouse carefully to avoid dehydration and hyperthermia.</li>
		<li>As soon as the LVs are dilated and visible, transfer the mouse to the restrainer and insert the plug far enough into the barrel to restrain movement. Monitor the mouse for normal activity such as breathing. Adjust the plug of restrainer if needed.</li>
		<li>Disinfect the injection area 3/4 from the tip of the tail with an alcohol wipe. Use a 27-30 G needle for mouse tail vein injection. Hold the tail firmly with one hand and insert the needle into the tail vein. Apply pressure on the syringe and complete the injection within 5-7 sec.</li>
		<li>Remove the needle from the tail vein and apply pressure with a cotton ball on the injection site to stop bleeding. Monitor the mouse for 15-30 min, then transfer the mouse back to vivarium once the breathing rate is reduced to its normal rate.</li>
	</ol>
	</li>
	<li>Confirmation of Systemic Mouse sRANKL Expression and Quantification of Mouse Serum TRACP5b Post Gene Transfer
	<ol>
		<li>Warm up the mouse in a cage for 5-10 min in order to dilate blood vessels and make LVs visible. Monitor the mouse carefully to avoid dehydration and hyperthermia.</li>
		<li>Make a small incision with a blade to the mouse's tail at a site one forth from the tip of the tail.</li>
		<li>Collect approximately 100 &#956;l of blood in serum separation tubes.</li>
		<li>Incubate serum separation tubes at room temperature for 30 min.</li>
		<li>Centrifuge samples at 10,000 x g for 5 min and collect serum. Store serum in -80 &#176;C for further analysis.</li>
		<li>Perform sRANKL ELISA and mouse serum TRACP5b assay on serum samples using a commercial available kit.</li>
	</ol>
	</li>
</ol>
2. In vitro Osteoclast Generation from Mouse BMMs
<ol>
	<li>Isolation and Culturing of BMMs
	<ol>
		<li>Isolation of the tibia and femur bones: Euthanize the donor mouse with CO2, and disinfect the legs with 70% ethanol. Dissect from the pubic bone to the calcaneus bone, to remove the femur and tibia bones intact.</li>
		<li>Remove the skin and muscle carefully without damaging the bone. Place processed femur and tibia bone in a Petri dish containing phosphate buffered saline (PBS).</li>
		<li>Removal of bone marrow: Cut off the tip on one side of the femur and tibia bones and flush out the bone marrow into a 50 ml tube using a 1ml syringe with a 25 G needle, loaded with &#945;-MEM containing 1% penicillin/streptomycin (Osteoclast Culture Medium/OCM).</li>
		<li>Process bone marrow: Mix and transfer bone marrow suspension to a new 50 ml tube by passing it through a 70 &#956;m nylon cell strainer. Centrifuge the cells at 300 x g and re-suspend in &#945;-MEM containing 1% penicillin/streptomycin and 10% FBS (Osteoclast Culture medium/OCM (+)).</li>
	</ol>
	</li>
	<li>Culture of Macrophages
	<ol>
		<li>Count cells using a hemocytometer or automatic cell counter.</li>
		<li>Plate 1 x 106 cells per well for a 6-well plate or 3 x 105 cells to glass coverslips (5 mm) or dentine slices placed in 96-well plate and incubate at 37 &#176;C.</li>
		<li>Aspirate all media containing non-adherent cells and incubate cells at 37 &#176;C with fresh OCM (+) containing 25 ng/ml mouse M-CSF for 48 hr.</li>
		<li>Aspirate all media and incubate cells at 37 &#176;C with fresh OCM (+) media containing 25 ng/ml mouse M-CSF, and 30 ng/ml mouse sRANKL to initiate osteoclastogenesis. Replenish media every 3 days as required.</li>
		<li>Grow cells for approximately 6-8 days to form giant multi-nuclear cells capable of resorbing bone. As soon as multinucleated cells appear, perform TRAP (tartrate resistant acid phosphatase) staining using a commercial available kit, and when fully matured, perform functional assays. Visualize F-actin rings on coverslips using phalloidin stain and image the cells attached to dentine slices by scanning electron microscopy (SEM) as described previously19.</li>
	</ol>
	</li>
</ol>
3. In vitro Osteoclast Generation from Human PBMCs
<ol>
	<li>Isolation of Human PBMCs
	<ol>
		<li>Add 10 ml of pre-warmed Histopaque-1077 into a 50 ml tube.</li>
		<li>Empty leukocyte filter obtained from blood bank with sterile PBS into a 50 ml tube (1:1 ratio of blood with PBS). Layer the diluted blood over the histopaque solution very slowly, making sure that the blood does not mix with the histopaque.</li>
		<li>Centrifuge samples at 1000 x g for 20 min at 18 &#176;C. Following centrifugation, carefully isolate the white buffy coat layer containing the white blood cells and transfer to a new 50 ml tube.</li>
		<li>Dilute cells with PBS and centrifuge again at 650 x g for 5 min to collect the cells.</li>
		<li>Remove supernatant and resuspend cells in OCM (-) media and count cells using cell counter.</li>
	</ol>
	</li>
	<li>Plating and Culturing PBMCs
	<ol>
		<li>Resuspend the cells in OCM (+) media and plate 8 x 106 cells/ml per well in a 6-well plate or 1 x 106 cells/ml per well in 96-well plate. Plate cells on glass coverslips (5 mm) as required for immunofluorescence imaging as well as on bone slices suitable for bone resorption assays as previously described20.</li>
		<li>Change culture media, replenishing the cells with human M-CSF every 2-3 days or as required. Then proceed to differentiation step on day 5 by adding 30 ng/ml of human sRANKL along with 25 ng/ml human M-CSF in OCM (+) media to initiate osteoclastogenesis.</li>
		<li>Grow cells for approximately 14 to 21 days to form giant multi-nuclear cells. As soon as multinucleated cells appear, these cells can then be labeled for TRAP, and when fully matured, functional assays can be performed. F-actin rings can be visualized on coverslips using phalloidin stain. The cell morphology can be examined in cells attached to dentine slices by SEM.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Musculoskeletal conditions are leading causes of morbidity and disability and are comprised of over 150 diseases and syndromes; affecting approximately 90 million Americans today. Joint inflammation and bone destruction are predominant features of musculoskeletal conditions, including arthritis and osteoporosis. Osteoporosis is a condition that weakens bone integrity, often leading to fractures of the bone. Arthritis is a chronic, debilitating disease characterized by inflammation of the joints that become swollen, tender and stiff&#8211;restricting normal movement and can lead to disability. Although the pathogenesis of musculoskeletal diseases greatly differs from one another, bone destruction is the overarching common feature. Bone destruction in musculoskeletal conditions is primarily caused by an imbalance between bone resorption and bone formation21,22. One therapeutic approach is to attempt to block the capacity of these cells to destroy bone23. The techniques and procedures outlined here allow the study of osteoclasts both in vivo and in vitro.
The in vivo model described here uses minicircles, which are small episomal DNA vectors (~4kb) freed from bacterial backbone, that also serve as transgene carriers24. Minicircles express transgenes 10-1,000 fold higher then normal bacterial plasmids with a stable expression for up to several months. The smaller molecular size of minicircles contributes to more efficient transfection rate and substantial expression compare to normal bacterial plasmids24. Therefore, minicircles are now widely applied in pre-clinical gene transfer studies25,26. Hydrodynamic delivery is a well-developed method based on hydrodynamic pressure in the capillaries to deliver plasmid DNA into the whole body of a mouse. Hydrodynamic gene delivery to hepatocytes via tail vein injection in mice is well-established to express transgene by plasmid DNA6. In these studies, overexpressing mouse sRANKL in the in vivo mouse model is achieved by sRANKL MC and hydrodynamic delivery via tail vein injections. Mouse sRANKL ELISA can be used to detect serum levels of RANKL in mice. Significant elevation of mouse serum TRACP5b, a bone resorption marker27, is observed in sRANKL gene transfer mice. Notably, this technique is not limited to overexpression of sRANKL; it can also be further applied to overexpress other secreted proteins28. The primary technical difficulty in this procedure is the hydrodynamic delivery via the tail vein. Apart from regular tail vein injection, hydrodynamic delivery requires delivery of minicircles in large volumes of physiological solution into the mice within 5-7 sec. Thus, steady handling and injection techniques are essential for success with this procedure. Generating sRANKL transgenic mice is another approach for in vivo osteoclastogenesis. However, a high level of sRANKL can lead to embryonic lethality4 and generating transgenic mice is also expensive and time-consuming.
In OVX rodent model, both inflammatory cytokines such as TNF, IL-1 and IL-6 and the non-inflammatory cytokine sRANKL contribute to in vivo osteoclastogenesis and bone loss. sRANKL gene transfer model leads to overexpression of only the non-inflammatory cytokine sRANKL. Therefore, the sRANKL gene transfer model described here offers researchers a powerful tool to examine the contribution of the RANKL-RANK axis in bone remodeling in the absence of inflammation.
An in vitro osteoclast culture method, which involves co-culturing bone marrow cells and osteoblasts, was established in the late 1980s prior to the discovery of RANKL in late 1990&#8217;s29,30. Recombinant M-CSF and RANKL induce the osteoclast differentiation efficiently in vitro and the method has been previously described in other studies3,31. M-CSF is known to stimulate RANK expression in early osteoclast precursors32 and to act as a survival factor for osteoclast precursors and mature osteoclasts33. RANKL is essential for osteoclast differentiation and activation3. Here, the method of in vitro osteoclastogenesis is described. Successful culturing and differentiation of precursor cells to osteoclasts require sterile cell isolation and proper plating techniques as well as careful understanding of timed cell harvest. Calculated plating of the cells is crucial because osteoclast differentiation requires fusion of osteoclast precursor cells34 and an inadequate number of osteoclast precursor cells will not facilitate fusion and differentiation of precursor cells into osteoclasts. Similarly, an excess number of osteoclast precursor cells, leads to clump formation and eventual cell death. Removal of non-adherent cells 2&#160;hr&#160;after plating the cells can help maintain an optimal cell number. Lastly, the time of cell harvest is crucial as osteoclasts are terminally differentiated short-lived cells with a lifespan of approximately 48 hr35. Thus, cells should be harvested within 48 hr&#160;after the first observation of multinucleated cells. These cells can then be characterized by TRAP and F-actin staining by immunohistochemistry and bone resorption activity can be detected by scanning electron microscopy (SEM). These experimental approaches enable clear definition of osteoclasts in terms of their phenotype and function.
The in vivo and in vitro techniques described here will facilitate the development of osteoclast inhibitors currently in pre-clinical studies, leading to efficacious therapeutic strategies for the millions of osteoporosis and arthritis patients worldwide.

Musculoskeletal diseases affect millions of people in the United States and present severe consequences for national and local health systems1. These disorders are characterized by loss of bone and joint function that require extensive treatment and long periods of recovery. Commonly, a relative increase in the number and/or activity of osteoclasts, cells specialized to resorb bone, in osteoporosis and arthritis is observed2. Under physiological conditions the number and activity of osteoclasts is regulated by receptor activator of nuclear factor &#954;-B ligand (RANKL), which is produced by osteoblasts. Osteoprotegerin (OPG), a decoy receptor for RANKL is also produced by osteoblasts3. In vivo animal models that involve systemic overexpression of sRANKL, or deletion of OPG are very valuable in osteoporosis research; however, these methods require the generation of transgenic mice4,5. Here, a novel alternative method of overexpressing sRANKL for the study of musculoskeletal-related disorders is described. Specifically, minicircle (MC) DNA technology and hydrodynamic delivery methods were used to achieve gene transfer of sRANKL in vivo and overexpress mouse sRANKL systemically6.
This method is also complementary to other in vivo models of osteoporosis, such as hormonal modulation of osteoclasts following ovariectomy7 and dietary intervention by low-calcium diet8. These models are very useful to study different aspects of musculoskeletal-related disorders however they require surgical procedures and may take up to several months, at a significant cost9. Ovariectomized (OVX) rodent model is an experimental animal model where removal of ovaries leads to estrogen deficiency thereby mimicking human postmenopausal osteoporosis10. Human post-menopausal osteoporosis, a condition where estrogen deficiency leads to increased risk of bone fractures and osteoporosis affects approximately eight million women in the United States alone. Although the OVX model is useful for post-menopausal osteoporosis it offers limited advantages in studying osteoporosis in general. Estrogen suppresses bone loss, by inducing osteoclast and inhibiting osteoblast apoptosis, therefore in its absence an increased osteoclast activity is observed10-12. A RANKL-OPG ratio imbalance that favors bone resorption is also observed13. However, estrogen deficiency in vivo is also accompanied by decreased levels of transforming growth factor &#946; (TGF &#946;), increased interleukin-7 (IL-7) and TNF, IL-1 and IL-614,15. As these cytokines have known bone remodeling modulatory functions independent of the RANKL pathway, it is impossible to attribute any osteoclast activation solely to the RANKL-RANK axis. The model described in this paper enables researchers to study in vivo RANKL-RANK axis in osteoclastogenesis and bone loss without pro-inflammatory cytokines compared to OVX rodent models.
Additionally, in vitro osteoclastogenesis techniques are essential tools to study osteoclast activation for potential therapeutic treatments of musculoskeletal diseases. Previous studies have also shown that culturing mouse bone marrow derived macrophages (BMMs) with mouse macrophage colony-stimulating factor (M-CSF) and mouse sRANKL can lead to osteoclast differentiation3,16,17. Here, the protocols to generate multinucleated osteoclast-like cells from mouse bone marrow as well as from human peripheral blood mononuclear cells (PBMCs) in vitro18 are described. The cell-based assays required to define a mature terminally differentiated and fully functional osteoclast are also briefly described. These in vitro techniques complement the novel in vivo approach and together serve as powerful investigative tools to study osteoclast differentiation and activation. Using these systems, scientists are able to generate osteoclasts in vivo and in vitro and define the stimuli and signals required for their proliferation and activation as well as test the efficacy of pharmacological and biological inhibitors.

<table><tbody><tr><td>alpha-MEM</td><td>Life Technologies&amp;nbsp;</td><td>12561-056</td><td></td></tr><tr><td>Human M-CSF</td><td>Miltenyi Biotec</td><td>130-096-492</td><td></td></tr><tr><td>Mouse M-CSF</td><td>Miltenyi Biotec</td><td>130-094-643</td><td></td></tr><tr><td>Human RANK-Ligand - soluble</td><td>Miltenyi Biotec</td><td>130-094-631</td><td></td></tr><tr><td>Mouse RANK-Ligand - soluble</td><td>Miltenyi Biotec</td><td>130-094-076</td><td></td></tr><tr><td>Tailveiner Restrainer for mice</td><td>Braintree</td><td>TV-150 STD</td><td></td></tr><tr><td>Mouse TRANCE/RANK L/TNFSF11 Quantikine ELISA Kit&amp;nbsp;</td><td>R&amp;amp;D systems</td><td>MTR00</td><td></td></tr><tr><td>Acid Phosphatase, Leukocyte (TRAP) Kit</td><td>Sigma</td><td>387A</td><td></td></tr><tr><td>MouseTRAP assay&amp;nbsp;</td><td>immunodiagnostic systems</td><td>SB-TR103</td><td></td></tr></tbody></table>

a novel in vivo gene transfer technique and in vitro cell based assays for the study of bone loss in musculoskeletal disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described. In Figure 1, the representative results of successful gene transfer of GFP and mouse sRANKL MC in mice are shown. In Figure 2, the representative images of mouse bone marrow or human PBMC cell differentiation time course cultures of precursor cells to osteoclasts by bright field microscopy are shown. Figure 3, the representative images from three independent methods for characterizing mouse bone marrow derived osteoclasts are shown. The presence of mouse sRANKL activates the expression of TRAP (Figures 3A and B) and the formation of F-actin rings (Figures 3C and D). Scanning electron microscopy (SEM) reveals that mouse sRANKL induces the formation of giant cells capable of resorbing bone (Figures 3E and F). Figure 4, the same characterization methods on human PBMC derived osteoclasts are shown.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51810/51810fig1highres.jpg" width="500" /> 
Figure 1. sRANKL in vivo gene-transfer leads to increased osteoclastogenesis. Anterior view of A, B) mouse liver explants and C, D) whole body images of 12-week old C57BL/6 male mice injected with 5 &#956;g of GFP or sRANKL MC DNA. The arrow indicates GFP expression in vivo in GFP gene transfer mouse. RANKL gene transfer mice serve as no background fluorescence. Images captured by Maestro 2 Imager, one-day post gene-transfer (red and green denote background and GFP, respectively). E) Plasmid map of sRANKL MC-DNA construct and F) sRANKL level in the serum analyzed at 1, 5 and 11 days post GFP or sRANKL gene transfer. G) Mouse serum TRACP5b level analyzed 11 days after GFP or sRANKL gene transfer. Scale bars indicate 5 mm in A, B, and 10 mm in C, D.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51810/51810fig2highres.jpg" width="500" /> 
Figure 2. In vitro osteoclastogenesis in both mouse and human cell culture systems. Bright-field microscopy of mouse cells isolated from bone marrow and cultured in the presence of 25 ng/ml mouse M-CSF and 30 ng/ml mouse sRANKL showing a differentiation time course at days A), 2 B) 5, and C) 8. Bright-field microscopy of human cells isolated from peripheral blood and cultured in the presence of 25 ng/ml human M-CSF and 30 ng/ml human sRANKL showing a differentiation time course at days D), 4 E) 14, and F) 21. Scale bars indicate 20 &#956;m.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51810/51810fig3highres.jpg" width="500" /> 
Figure 3. Generation and characterization of mouse osteoclasts. TRAP staining (pink) of mouse cells isolated from bone marrow and cultured for 6 days in the presence of A) 25 ng/ml mouse M-CSF or B) 25 ng/ml mouse M-CSF and 30 ng/ml mouse sRANKL. Phalloidin (green) and DAPI (Blue) staining of mouse cells isolated from bone marrow and cultured for 8 days in the presence of C) 25 ng/ml mouse M-CSF or D) 25 ng/ml mouse M-CSF and 30 ng/ml mouse sRANKL showing the formation of F-actin ring in multinucleated cells. SEM photomicrographs of mouse cells isolated from bone marrow and cultured for 8 days in the presence of E) 25 ng/ml mouse M-CSF or F) 25 ng/ml mouse M-CSF and 30 ng/ml mouse sRANKL showing the formation of resorption areas on bone as indicated by the arrows. Scale bars indicate 40 &#956;m in A-D, and 30 &#956;m in E, F.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51810/51810fig4highres.jpg" width="500" /> 
Figure 4. Generation and characterization of human osteoclasts. TRAP staining (pink) of human cells isolated from PBMCs and cultured for 15 days in the presence of A) 25 ng/ml human M-CSF or B) 25 ng/ml human M-CSF and 30 ng/ml human sRANKL. Phalloidin (green) and DAPI (Blue) staining of human cells isolated from PBMC and cultured for 19 days in the presence of C) 25 ng/ml human M-CSF or D) 25 ng/ml human M-CSF and 30 ng/ml human sRANKL showing the formation of F-actin rings in multinucleated cells. SEM photomicrographs of human cells isolated from PBMC and cultured for 18 days in the presence of E) 25 ng/ml human M-CSF or F) 25 ng/ml human M-CSF and 30 ng/ml human sRANKL showing the formation of resorption areas on bone as indicated by the arrows. Scale bars indicate 10 &#956;m in A, B, 20 &#956;m in C, D 15 &#956;m in E, F.

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A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

11.6K Views.  University of California, Davis. Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described. 

Video: A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Developmental Biology

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

Cancer Research

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Bioengineering

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Immunology and Infection

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (&#181;CT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent &#181;CT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ...

Osteo-Organoid-Derived Cells for Effective HSCT in Irradiated Mice

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

Multimodal Analysis of Bone Formation Using Explant Models

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

Author Spotlight: Advanced Techniques for Characterizing Tissue Mineralization in Bone Regeneration Research

The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical investigations.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

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