This project was funded by grants from the Singapore Ministry of Education (MOE, grant number 2013-T2-2-126) and the National Institute of Health, USA (NIH, grant number 1R21AT008452-01A1). T.Y. received a graduate scholarship from the NUS Department of Biological Sciences. We thank the confocal unit of the NUS Centre for Bioimaging Sciences (CBIS) for their constant support.

All experiments were performed in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols of the National University of Singapore (R14-293).
1. Fish Husbandry and the Collection of Embryos
<ol>
	<li>Raise WT, ctsk:nlGFP24, RANKL:HSE:CFP24, and osx:mCherry21 single- or compound-transgenic medaka fish at 26 &#176;C under a controlled light cycle (14 h light, 10 h dark) to induce spawning.</li>
	<li>Daily spawning takes place during the first 30 min after the light is switched on. The eggs stick together through filaments and attach to the female&#39;s abdomen for several hours. Use a fine-meshed net to catch a female adult carrying an egg cluster. Let the fish briefly rest in the net and then gently massage the fish&#8217;s abdomen to carefully strip the fertilized egg cluster from the abdomen of the female. 
	NOTE: A healthy medaka female can produce 10 - 20 eggs each day for approximately 5 months.</li>
	<li>Place the eggs into a 60&#160;mm plastic Petri dish. Use a plastic pipette to rinse the embryos with 5 - 10 mL of 0.3x Danieau&#39;s solution (fish medium; 19.3 mM NaCl, 0.23 mM KCl, 0.13 mM MgSO4, 0.2 mM Ca(NO3)2, and 1.7 mM HEPES,&#160;pH 7.0). Add 1 mL of a 0.25% (w/v) methylene blue stock solution to 2.5 L of fish medium to prevent fungal growth.</li>
	<li>Gently roll egg cluster to form a knot of attachment filaments. Use forceps to carefully remove the attachment filaments from the fertilized egg clusters to obtain individual embryos&#160;(Figure&#160;1A).</li>
	<li>Stage the embryos according to Iwamatsu 200413.</li>
	<li>Culture 20 - 30 embryos per 60&#160;mm plastic Petri dish in a 28 &#176;C incubator. Change the medium daily to ensure normal development of the embryos. 
	NOTE: The time around the hatching stage (8 - 9 d postfertilization, DPF) is especially critical for survival. Remove free-floating chorions to keep the medium clean and to ensure good larval survival rates.</li>
</ol>
2. Transgenic Embryo Screening
<ol>
	<li>Use a stereomicroscope equipped with a mercury lamp for fluorescence imaging and GFP, RFP, and CFP filters to screen transgenic embryos for fluorescent reporter expression using 40X magnification.</li>
	<li>Visually identify osx:mCherry embryos by mCherry reporter expression in the early-forming cranial bones, such as the cleithrum, on both sides of the posterior head (Figure 1B, arrow), and the parasphenoid, at a central position in the ventral cranium (Figure 1B, arrowhead). 
	NOTE: Reporter expression starts from 5 DPF onwards21.</li>
	<li>Identify ctsk:nlGFP embryos by strong nlGFP expression in the head (Figure 1C, arrow) and tail (Figure 1C, arrowhead), starting from 6 DPF. 
	NOTE: Endogenous osteoclasts only form after 21 DPF. nlGFP-expressing cells at this early stage (6 DPF) are not osteoclasts but other, so far uncharacterized, ctsk-positive cells24.</li>
	<li>Identify RANKL:HSE:CFP transgenic embryos by ubiquitous CFP expression after a short heat&#160;shock treatment for 20 min at 39 &#176;C, conducted at 2 DPF or later for screening purposes. 
	NOTE: The RANKL and CFP transgenes are under the control of the same bidirectional Heat&#160;Shock Element (HSE). CFP expression indicates successful RANKL induction24.</li>
	<li>Perform a 1.5 - 2&#160;h heat&#160;shock treatment at 9 DPF or later to induce large numbers of ectopic osteoclasts in the trunk region, which consequently results in an osteoporosis-like phenotype24. 
	NOTE: Transgenic RANKL expression induced at 9 DPF results in an ectopic activation of dormant osteoclast progenitor cells, which endogenously is not triggered before 21 DPF. Use a water bath to obtain stable 39 &#176;C conditions. Let the Petri dish containing medaka embryos float on the water surface. Make sure the lid of the petri dish is dry to prevent the sinking of the dish.</li>
	<li>Screen embryos from compound lines, such as RANKL:HSE:CFP/ctsk:nlGFP double-transgenic and osx:mCherry/RANKL:HSE:CFP/ctsk:nlGFP triple-transgenic, according to the expression pattern of each individual transgene. 
	NOTE: Hemizygous and homozygous transgenic embryos were distinguished by different fluorescence levels of the reporter transgene. Homozygous embryos had a fluorescence intensity that was approximately doubled compared to that of hemizygous transgenics. Compound lines that were homozygous for both RANKL:HSE:CFP and ctsk:nlGFP were obtained by repeated incrossing over several generations. For triple-transgenic osx:mCherry/RANKL:HSE:CFP/ctsk:nlGFP fish, homozygous RANKL:HSE:CFP/ctsk:nlGFP fish were crossed with homozygous osx:mCherry carriers. The resulting heterozygous triple-transgenic progeny were raised and incrossed to obtain embryos homozygous for RANKL:HSE:CFP. The RANKL:HSE:CFP transgene must be homozygous in order to obtain the efficient induction of ectopic osteoclasts.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55025/55025fig1.jpg" />
Figure 1: WT and Transgenic Medaka Embryos at 7 D&#160;Postfertilization (DPF).&#160;A. WT embryos observed with brightfield illumination.&#160;B. Transgenic embryos showing&#160;osx:mCherry expression around the cleithrum (arrow) and parasphenoid (arrowhead).&#160;C. Transgenic embryos showing&#160;ctsk:nlGFP expression in the head (arrow) and tail (arrowhead). Scale bars: 500 &#181;m.&#160;<a href="http://cloudfront.jove.com/files/ftp_upload/55025/55025fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Bisphosphonate Treatment of Medaka Larvae
<ol>
	<li>Prepare solutions containing different concentrations of bisphosphonates (BPs) for dose-response studies. 
	NOTE: The exemplary BP used in this protocol is alendronate.
	<ol>
		<li>Dissolve alendronate in fish medium at a concentration of 100 &#181;g/mL to prepare a stock solution.</li>
		<li>Use a vortex mixer to ensure complete dissolution. Store the stock solution at 4 &#176;C.</li>
		<li>Prepare different working solutions by diluting the stock solution with fish medium to a series of concentrations (i.e., 25, 37.5, 50, 62.5, and 75 &#181;g/mL). 
		NOTE: Different drugs may have different Absorption, Distribution, Metabolism, and Excretion (ADME) parameters, which must be considered during testing using this medaka larvae system. Also, drug solubility and stability may vary when applied as an aqueous solution. Less water-soluble compounds might need to first be dissolved in organic solvents, such as DMSO. In this case, a stock solution is prepared in DMSO, which is then further diluted in fish medium. Note that the working solutions in water (fish medium) can be stored in the refrigerator for several wk. However, solutions containing DMSO must be stored at RT to prevent crystallization.</li>
	</ol>
	</li>
	<li>Transfer medaka larvae into six-well plates (six larvae/well) for subsequent BP (alendronate) treatment.</li>
	<li>Remove the fish medium carefully using a clean plastic pipette and add a small volume (approx. 0.5 mL) of alendronate solution to each well.</li>
	<li>Avoid leftover fish medium, as the added BP solution might be diluted, which is especially critical for less-concentrated alendronate solutions.</li>
	<li>Remove a small volume of alendronate solution (up to 0.5 mL) from each well with a clean plastic pipette and replace it with a larger volume (4 mL) of alendronate solution.</li>
	<li>Change medium daily to ensure normal&#160;embryos development.</li>
</ol>
4. Live Staining of Mineralized-bone Matrix
<ol>
	<li>Dissolve 0.5 g of Alizarin complexone (ALC; alizarin-3-methyliminodiacetic acid) or 0.05 g of calcein in 50 mL of fish medium to prepare 1% and 0.1% stock solutions, respectively. Use a vortex mixer to ensure complete dissolution. 
	NOTE: Fish medium without the addition of methylene blue is used in this and the subsequent steps to reduce autofluorescence in the larvae.</li>
	<li>Use a syringe and single-use filter (0.2 &#181;m) to filter the staining solution. Store the filtered solution in the dark at RT. 
	NOTE: The color of the filtered, clear ALC staining solution is dark yellow to orange. The color of the filtered, clear calcein solution is bright yellow. Solutions can be used for several months.</li>
	<li>Dilute the filtered ALC or calcein stock solution 1:10 in fish medium and incubate the medaka larvae for 1.5 - 2 h (0.1% ALC solution) or 2 - 2.5 h (0.01% calcein solution) in a 28 &#176;C incubator if larvae between 9 and 17 DPF are used. Keep the samples in the dark.</li>
	<li>Transfer the larvae to fresh fish medium using a clean plastic pipette.</li>
	<li>Remove the fish medium with a clean plastic pipette and add fresh fish medium. Repeat this step for 3 - 4 times until no red- or yellow-stained solution (ALC or calcein, respectively) is left over. Leave the larvae in fish medium for 30 - 60 min&#160;before mounting them for imaging to avoid epifluorescence from the medium. 
	NOTE: 0.1% ALC staining solution is harmful to medaka larvae for extended exposure times. Incubation times of longer than 2 h affect the survival of the larvae. Concentration and staining time therefore need to be optimized for different stages in order to achieve optimal embryo survival and staining results.</li>
</ol>
5. Live Fluorescence Imaging
<ol>
	<li>Anesthetize the medaka larvae with 0.01% Tricaine (ethyl 3-aminobenzoate methanesulfonate) in fish medium. 
	NOTE: Anesthetized larvae become immobilized after 5 - 10 min in Tricaine solution and usually are lying either on their sides or their backs.</li>
	<li>Use a plastic microloader to orientate the larvae according to the region of interest. The orientation of the larvae used in this protocol is lateral.</li>
	<li>Use a stereomicroscope with fluorescence illumination for imaging. Use high magnification when taking images, focusing on different parts of the larvae (head, anterior trunk, posterior trunk, and tail). Stitch individual images together at overlapping regions using a suitable image-processing software (insets in Figure&#160;3G). 
	NOTE: This helps to improve the image quality of all relevant body parts in the correct focal plane.</li>
	<li>Return the larvae to the fish medium for recovery after imaging.</li>
</ol>
6. Live Confocal Imaging
<ol>
	<li>Anesthetize the larvae with 0.01% Tricaine in fish medium for 5 - 10 min until they become immobilized.</li>
	<li>Dissolve low-melting agarose to 1.5% in fish medium by heating it in a microwave oven. Cool this solution to approximately 30 &#176;C.</li>
	<li>Add 0.5 - 1 mL of liquid 1.5% low-melting agarose in fish medium to a glass-bottomed Petri dish. Transfer the anesthetized larvae into the solution using a clean plastic pipette. 
	NOTE: Take special precaution that the temperature of the liquid low-melting agarose is low enough to not harm the larvae.</li>
	<li>Before the agarose solidifies, use a plastic microloader to push the larvae to the bottom of the Petri dish and orientate the larvae according to the region of interest. The orientation of the larvae used in this protocol is lateral. 
	NOTE: The samples are ready for confocal live imaging after the agarose completely solidifies.</li>
	<li>Use a confocal microscope to acquire images.</li>
	<li>Use a 543 nm laser line for mCherry and ALC staining analyses. Use a 488&#160;nm laser line for nlGFP and calcein staining analyses.</li>
	<li>After imaging, add fish medium to the Petri dish and use a pair of fine syringe needles (27 G x 1&#189;&#34;) to carefully remove the larvae from the agarose. Transfer the larvae with residually attached agarose to a Petri dish with fish medium to recover.</li>
	<li>Process the images using an image analysis software27.</li>
</ol>

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The authors declare that they have no competing or financial interests.

Critical Steps within the Protocol
It is essential that the conditions for heat&#160;shock treatment are consistent and stable when comparing different samples. Stable temperature conditions guarantee similar levels of RANKL induction in the transgenic larvae and, consequently, comparable osteoclast formation, which can be confirmed by screening for ctsk:nlGFP expression. Ultimately, this leads to a similar degree of induced ectopic bone resorption and osteoporosis-like lesions, as valid...

The vertebrate skeleton provides structural support and protection for organs, allows mobility, and serves as a source of calcium. Throughout life, the extracellular bone matrix is continuously turned over to maintain bone stability and rigidity. This process requires the tightly coordinated activity and interplay of bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts are derived from multipotent mesenchymal progenitors and produce collagen to form the osteoid, the proteinaceous part of the bone matrix10. Osteoblasts interact with osteoclasts to achieve a balanced activity of both cell types, which is required to control bone homeostasis7. Because of these intricate regulatory interactions, responses to drug treatment and bone homeostasis cannot be fully examined using in vitro studies. Hence, there is a strong demand for animal models. Compared to the cell&#160;culture settings, in vivo models can provide valuable insight into the multicellular networks within the bone environment.
Numerous mouse models exist for a variety of human bone disorders including osteoporosis16. However, the size and accessibility of mouse embryos represent significant limitations for live imaging of skeletal processes. Small teleost fish, on the other hand, serve as an attractive alternative for in vivo imaging. Zebrafish (Danio rerio) and medaka (Oryzias latipes) have become popular animal models for skeletal research over the last two decades17, 19, 22, 24. Bone in teleost fish and in mammals is very similar, both on a structural and on a physiological level, and many of the key regulatory genes and signaling pathways are conserved3. As in mammals, teleost fish carefully regulate the activity of osteoblasts and osteoclasts to balance bone formation and resorption26. Most importantly, the optical clarity of fish larvae allows the use of fluorescent reporters to label bone cells and the calcified skeletal matrix8, 9, 12, 21, 23, which&#160;facilitates the observation of cellular processes in the living animal. In addition, a series of genetic tools has been generated to facilitate biomedically relevant research in fish. For medaka in particular, methods for targeted gene mutation by CrispR/Cas92, cell-lineage tracing6, and site-specific transgenesis14 have been recently established and are now widely in use15.
Small teleost larvae have been successfully used for chemical screens, which led to the discovery of several pharmacologically relevant drugs1, 18.
Fish larvae are tolerant to low concentrations of DMSO and are able to absorb compounds from their aquatic environment, either through the skin or through the gastrointestinal tract1, 5. Our lab previously reported transgenic medaka lines that express fluorescent reporters in bone cells under the control of various osteoblast- and osteoclast-specific promoters. These include premature osteoblasts (collagen 10a1, col10a1; osterix, osx)20, 21, mature osteoblasts (osteocalcin, osc)27, and osteoclasts (cathepsin K, ctsk)24. We also generated a transgenic line that expresses the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter24.
Induction of RANKL in this system results in the ectopic formation of active osteoclasts. This leads to increased bone resorption and a severe osteoporosis-like phenotype, with drastically reduced mineralization in the vertebral bodies. We recently showed that osteoclast activity in this model can be blocked by the bisphosphonates etidronate and alendronate, two drugs commonly used in human osteoporosis therapy, thus validating medaka as a suitable model system for osteoporosis27.
Due to their large brood size, rapid development, and small size of embryos, transgenic medaka larvae are uniquely suited for the large-scale screening of osteoporosis drugs and for the in vivo analysis of bone cell behavior. Studies in medaka thus can efficiently complement experiments in cell cultures and in mice that are aimed at discovering new therapeutic targets and novel therapies for human bone disorders.
In the present study, we describe a protocol to treat medaka bone-reporter larvae with the common osteoporosis drug, alendronate. We also describe in detail how treated larvae are mounted and prepared for the live imaging of bone matrix and bone cells. These protocols can be easily adapted to other small chemical compounds that either work as bone anabolic or antiresorptive drugs.

<table><tbody><tr><td>Alendronate&amp;nbsp;</td><td>Sigma</td><td>A4978</td><td></td></tr><tr><td>alizarin-3-methyliminodiacetic acid, Alizarin Complexone</td><td>Sigma</td><td>A3882</td><td></td></tr><tr><td>Calcein</td><td>Sigma</td><td>C0875</td><td></td></tr><tr><td>ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td><td>Sigma</td><td>A5040</td><td></td></tr><tr><td>ImageJ (1.4.3.67)</td><td>National Institute of Health (NIH)</td><td></td><td>https://imagej.nih.gov/ij/</td></tr><tr><td>LSM 510 Meta confocal&amp;nbsp;</td><td>Zeiss</td><td></td><td></td></tr><tr><td>LSM Image Browser (4.2.0.121)</td><td>Zeiss</td><td></td><td>http://www.zeiss.com/microscopy/en_de/downloads/lsm-5-series.html</td></tr><tr><td>Micro-loader</td><td>Eppendorf</td><td>5242956003</td><td>Eppendorf ep T.I.P.S 20 &amp;mu;L</td></tr><tr><td>NIS-Elements BR 3.0 software</td><td>Nikon</td><td></td><td></td></tr><tr><td>Photoshop CS6 (13.0.0.0)</td><td>Adobe</td><td></td><td></td></tr><tr><td>SMZ1000 stereomicroscope&amp;nbsp;</td><td>Nikon</td><td></td><td></td></tr></tbody></table>

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.

Abundant egg numbers, as well as the small size of the larvae, make medaka an excellent model for drug screening. A single six-well plate was used to culture up to 36 larvae, which was sufficient to provide statistically significant data. Another big advantage of using fish for skeletal analysis is the possibility of doing live imaging. The transparency of fish larvae allows the use of fluorescent proteins to label bone cells, as well as the use of dyes that bind to bone matrix in order to visualize mineralization. Fish ...

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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.

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 ...

drug-treatment-vivo-imaging-osteoblast-osteoclast-interactions-medaka

Research

JoVE Journal

Developmental Biology

9.2K Views.  National University of Singapore. 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.

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

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

Cancer Research

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

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 ...

Medicine

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

Bioengineering

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.

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.

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

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 ...

Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the specific effects and mechanisms in zebrafish remain unclear. Alizarin red has a high affinity for calcium ions and can help visualize the bone and illustrate skeletal mineral mass. In this study, we aimed to detect lead acetate (PbAc)-induced bone loss in zebrafish larvae by using alizarin red staining. Zebrafish embryos were treated with a series of PbAc concentrations (0, 5, 10, 20 mg/L) between 2 and 120 h post fertilization. Whole-mount skeletal staining was conducted on larvae at 9 days post fertilization, and the total stained area was quantified using ImageJ software. The results indicated that the mineralized tissues were stained in red, and the stained area decreased significantly in the PbAc-exposure group, with a dose-dependent change in bone mineralization. This paper presents a staining protocol for investigating skeletal changes in PbAc-induced bone defects. The method can also be used in zebrafish larvae for the detection of bone loss induced by other chemicals.

Here, we have used alizarin red staining to show that lead acetate exposure causes a bone mass change in zebrafish larvae. This staining method can be adapted to the investigation of bone loss in zebrafish larvae loss induced by other hazardous toxicants.

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the ...

Laser Ablation Method to Study Bone Regeneration in Zebrafish Larvae

Two-Photon Laser-Mediated Ablation of Osteoblasts in Developing Zebrafish Larvae

Zebrafish (Danio rerio) have an outstanding capacity to regenerate different organs and appendages. Bone regeneration in zebrafish has been studied using different methods such as fin amputation, scale plucking, skull trepanation, and microscopic approaches. Using a confocal laser scanning setup equipped with a two-photon laser, a laser ablation method was developed as a lesion paradigm to ablate bone-forming cells (osteoblasts) in the developing opercle of zebrafish larvae. The method described here allows the ablation of cells in a precise manner, as the area, shape, and depth can be finely adjusted. In addition, this method allows imaging of the area before and just after the ablation, so that short-term effects of the injury can be analyzed. In this experimental setup, the immune response after ablation of osteoblasts in the injured area was studied. An increase in the recruitment of macrophages was observed after ablation, indicating the relevance of their presence during bone regeneration.

Author Spotlight: Immune-Mediated Bone Regeneration &#8212; The Critical Role of Macrophages and Controlled Immunomodulation in Restorative Processes

This protocol describes a two-photon laser ablation approach carried out in zebrafish larvae, which serves as a model to study bone regeneration and the effects of the immune response to ablation.

Zebrafish (Danio rerio) have an outstanding capacity to regenerate different organs and appendages. Bone regeneration in zebrafish has been studied using ...

A Fluorescent Intravital Imaging Approach to Study Load-Induced Calcium Signaling Dynamics in Mouse Osteocytes

Bone tissue is exquisitely sensitive to differences in mechanical load magnitude. Osteocytes, dendritic cells that form a syncytium throughout the bone, are responsible for the mechanosensory function of bone tissue. Studies employing histology, mathematical modeling, cell culture, and ex vivo bone organ cultures have greatly advanced the understanding of osteocyte mechanobiology. However, the fundamental question of how osteocytes respond to and encode mechanical information at the molecular level in vivo is not well understood. Intracellular calcium concentration fluctuations in osteocytes offer a useful target for learning more about acute bone mechanotransduction mechanisms. Here, we report a method for studying osteocyte mechanobiology in vivo, combining a mouse strain with a fluorescently genetically encoded calcium indicator expressed in osteocytes with an in vivo loading and imaging system to directly detect osteocyte calcium levels during loading. This is achieved with a three-point bending device that can deliver well-defined mechanical loads to the third metatarsal of living mice while simultaneously monitoring fluorescently indicated calcium responses of osteocytes using two-photon microscopy. This technique allows for direct in vivo observation of osteocyte calcium signaling events in response to whole bone loading and is useful in the endeavor to reveal mechanisms in osteocyte mechanobiology.

The current article describes performing an intravital imaging approach to observe mechanically induced calcium signaling of embedded osteocytes in vivo in real-time in response to tissue-level mechanical loading of the mouse third metatarsal.

Bone tissue is exquisitely sensitive to differences in mechanical load magnitude. Osteocytes, dendritic cells that form a syncytium throughout the bone, ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

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

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