This work was supported in part by a JSPS KAKENHI grant from the Japan Society for the Promotion of Science (No. 19K10397 to H.K. and No. 18K09862 to I.M.).

All animal procedures and animal care were performed in accordance with Tohoku University rules and regulations.
1. Dissection of newborn dmp1-topaz mouse calvaria
<ol>
	<li>For this protocol, use 6&#8210;7 day old pups of C57BL/6-Tg(Dmp1-Topaz)1Ikal/J mice. Euthanize mice with 5% isoflurane inhalation and then transfer the pups into 70% ethanol.</li>
	<li>Transfer the euthanized pup into a non-treated culture dish.</li>
	<li>Using scissors and tweezers, grab the skin at the base of the skull and make an incision.</li>
	<li>Using the first incision as a starting point, cut on both lateral sides of the skull in front of the ears, remove the skin, and expose the calvaria.</li>
	<li>Hold the head from the nasal bridge and cut the calvaria along the lambdoid suture. Cut along the lateral edges of the parietal bones and extend it to include the frontal part.</li>
	<li>Separate the calvaria from the underlying brain tissue. 
	NOTE: To ensure a clean bony calvaria with minimal soft tissue attachment do not cut deep into the bone; for reference, one should be able to see the shadow of the scissors beneath the bone.</li>
	<li>Transfer the calvaria into phosphate-buffered saline (PBS, pH 7.4 throughout the protocol). Make sure the concave part is facing upwards.</li>
</ol>
2. Fractionation of newborn mouse calvaria
<ol>
	<li>Transfer up to 5 calvariae to a 50 mL conical tube containing 5 mL of 2 mg/mL collagenase solution. Use fresh collagenase solution prepared just before use.
	<ol>
		<li>To prepare a fresh collagenase solution, add collagenase powder to isolation buffer (70 mM NaCl, 10 mM NaHCO3, 60 mM sorbitol, 3 mM K2HPO4, 1 mM CaCl2, 1 mg/mL bovine serum albumin (BSA), 5 mg/mL glucose and 25 mM HEPES) at 2 mg/mL. Dissolve the collagenase powder on a magnetic stirrer.</li>
		<li>Run the collagenase solution through a 0.22 &#956;m sterile filter unit into a 50 mL tube and keep it on ice throughout the protocol.</li>
	</ol>
	</li>
	<li>Incubate the calvaria at 37 &#176;C for 20 min on a shaker at 300 rpm to obtain fraction 1.</li>
	<li>Discard the digest of fraction 1, wash the calvaria with 5 mL of phosphate buffered saline (PBS) and discard the wash.</li>
	<li>Incubate the calvaria in 5 mL of 5 mM ethylenediaminetetraacetic acid (EDTA) containing 1 mg/mL BSA in PBS, PH 7.4 at 37 &#176;C for 15 min on a shaker at 300 rpm.</li>
	<li>Collect the digest in a 50 mL conical tube. Wash the calvaria with 5 mL of PBS and add the wash solution to the digest. 
	NOTE: At each point of collecting the digest and washing, pipette the bones in their solution to detach the cells from the bone and to prevent doublets and cell aggregates.</li>
	<li>To obtain fraction 2, centrifuge the digest at 300 x g at 4 &#176;C for 5 min. Discard the supernatant, re-suspend the pellet in 8 mL of &#945;-Minimum Essential Medium (MEM) containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin G, and 100 &#956;g/mL streptomycin. Seed on a 10 cm culture dish. Incubate fraction 2 at 37 &#176;C under 5% CO2.</li>
	<li>Incubate the calvaria in 5 mL of collagenase solution at 37 &#176;C for 20 min on a shaker at 300 rpm.</li>
	<li>To collect fraction 3, repeat steps 2.5&#8210;2.6. Incubate fraction 3 at 37 &#176;C under 5% CO2. 
	NOTE: At this point, the bones will become smaller, and they will be reduced to fragments. To ensure that no bones get accidentally aspirated into the digests, which leads to cell loss in the process, use a small tip pipette for collecting the digests (1000&#8210;200 &#956;L pipette tips).</li>
	<li>Incubate the calvaria in 5 mL of collagenase solution at 37 &#176;C for 20 min on a shaker at 300 rpm.</li>
	<li>To collect fraction 4, repeat steps 2.5&#8210;2.6. Incubate fraction 4 at 37 &#176;C under 5% CO2.</li>
	<li>Incubate the calvaria in 5 mL of 5 mM EDTA containing 1 mg/mL BSA in PBS (pH 7.4) at 37 &#176;C for 15 min on a shaker at 300 rpm.</li>
	<li>To collect fraction 5, repeat steps 2.5&#8210;2.6. Incubate fraction 5 at 37 &#176;C under 5% CO2. Osteocytes can be prepared for sorting on the same day or cultured up to 24 h before sorting.</li>
</ol>
3. Preparation of osteocytes for fluorescence activated cell sorting
<ol>
	<li>Aspirate the medium of each cell fraction and wash gently with 10 mL of PBS twice.</li>
	<li>Add 5 mL of 0.5% Trypsin-EDTA in PBS and incubate the cells for 5 min at 37 &#176;C. Add 5 mL of 10% FBS &#945;-MEM and pipette to detach the cells gently. Combine the cells in each fraction by sifting through a 40 &#956;m cell strainer into a 50 mL conical centrifuge tube.</li>
	<li>Wash the cells with 10 mL of PBS and collect by sifting through a 40 &#956;m cell strainer into a 50 mL conical centrifuge tube. Centrifuge the cells at 300 x g at 4 &#176;C for 5 min.</li>
	<li>Aspirate the supernatant and add 10% FBS &#945;-MEM to the pellet. Adjust cell concentration to approximately 1 x 107 cells/mL.</li>
	<li>Filter the cells in a 35 &#956;m nylon mesh capped tube. The cells are now ready for FACS.</li>
	<li>For this protocol, use a 100 &#956;m size nozzle. The collection fluid should consist of 10% FBS &#945;-MEM. Keep the sample being sorted on agitation and at 4 &#176;C throughout the sort if possible.</li>
	<li>Prior to the sort, optimize gating to remove artifacts and cells by adjusting the side scatter (SSC) area and forward scatter (FSC) area. Eliminate doublets by adjusting SSC-width vs SSC-Height and FSC-area vs FSC-width. GFP-negative osteocytes should be used as a control to adjust the parameters of the sort instrument.</li>
	<li>After cell collection is complete, centrifuge the cells at 300 x g at 4 &#176;C for 10 min. Wash cells with PBS. Centrifuge the cells at 300 x g at 4 &#176;C for 5 min.</li>
	<li>Re-suspend the cells and adjust the number as desired using 10% FBS &#945;-MEM.</li>
</ol>

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The authors have nothing to disclose.

The first isolated osteocyte was from a chicken calvaria7 isolated by using (OB7.3) or the aviant variant of PHEX; however, this method is limited by the availability of workable antibodies, as osteocyte-specific antibodies that are also specie-specific have to be manufactured. Researchers used a different modification of the sequential enzymatic process to obtain osteocytes from mouse and rat long bones; the reported purity of these cultures were set at about 70%9. The development of the cre mouse model allowed for engineering osteocytes, which express GFP on their surface. This mouse model, along with FACS, was used to obtain pure cultures of primary osteocytes20.
We omit the use of fractions 1 and fractions 6&#8210;8 as little amounts of osteocytes come off in these fractions. Using fractions 2&#8210;5 give the best possible yield of osteocytes over the shortest processing time; this limits the handling time of osteocytes and works towards preventing cell death or a possible alteration in cell signaling as a result of the stress the cell is subjected to during fractionation. We also culture osteocytes for 24 h pre-sort, which by default, excludes non-adherent suspension cells (hematopoietic cells) during sort preparation. This minimizes contamination by hematopoietic cells expressing GFP22. The workflow provided in this protocol takes advantage of previously published methods23 and shortens the time of the sort allowing for an efficient and quick osteocyte recovery with minimal contamination.
Critical steps in the protocol include obtaining a clean bony calvaria and trimming soft tissue from the brain or connective tissue attached to the bone to limit the contamination of adherent cells (fibroblast and neural cells). Also, pipetting the cells during steps that include obtaining and washing the digests is crucial, since cell aggregates and doublets are read as waste during sorting and will contribute to a low yield of osteocytes.
Osteocytes can be sorted without prior culture. However, this means that a higher number of cells have to be sorted, increasing the time of the sort and increasing the chance of hematopoietic contamination. This can be mitigated by applying hematopoietic cell depletion pre-sort. However, this is taxing and not recommended for routine and batch laboratory analysis22. Trouble may arise while sorting due to the presence of large cell aggregates and doublets clogging fluid flow of the sort machine. In our protocol, this has not been an issue, but this can be solved by reducing the FBS content of the sort buffer (less than 10%).
This protocol does not come without limitations. This method utilizes mouse osteocytes, which do not entirely resemble human osteocytes. This restricts extending the results obtained by studying murine osteocytes to meaningful clinical outcomes. Protocols for isolation of human osteocytes have been described24, and researchers are encouraged to use the cell species that best serves their goals. As with other protocols, the quantities of osteocytes obtained using this protocol are limited, and a large number of mice are required for large scale analysis, however, by decreasing the time needed for preparing and sorting osteocytes, higher quantities of cells can be acquired in a single time frame.
Osteocytes obtained through this process can be used for further culture and co-culture, gene expression analysis, downstream analysis of substrate activation/ inhibition, molecular probing, and staining applications. Primary cells can also be used to construct a 3D osteocyte matrix model resembling the native osteocytic environment for the study of mechanotransduction and mechanosensing.

Osteocytes are terminally differentiated cells from osteoblastic progenitors that became embedded in their secreted matrix1. They are the most abundant and longest-living cells among bone cell populations. They reside within lacunae and have a characteristic stellate morphology with dendrites that extend through channels called canaliculi forming an extensive network of communication and metabolic exchange with their surrounding environment and the bone surface2. Osteocytes choreograph both osteoblasts and osteoclasts roles in bone remodeling, they are the primary mechanosensory cells conferring adaptation to mechanical loading3, are involved in phosphate homeostasis4 and bone matrix mineralization5, and together with the lacunocanalicular system, they act as an endocrine organ signaling distant tissues6.
Osteocytes are situated within a mineralized matrix, which limits accessibility and renders their isolation challenging, thus hindering in vitro investigation. One of the first isolation methods described isolated osteocytes from chicken calvaria using an osteocyte-specific monoclonal antibody (OB7.3)7 which was later known to be the avian variant of PHEX (PHosphate-regulating gene with homology to Endopeptidases on the X chromosome)8. Other researchers used sequential digestion of rat9 or mouse10 long bones to obtain osteocyte-rich fractions in which osteocyte purity was reported to be around 70%9. Limitations of this procedure include the sub-optimal purity of cultures contaminated with other cell types besides osteocytes and that osteocytes could be potentially overgrown by other cells in culture since osteocytes have lost the ability to divide. These challenges restrict the usability of long term cultures.
To overcome these limitations, different osteocyte cell lines were developed. The MLO-Y4 cell line11 and the MLO-A5 cell line12 are notably the most widely studied cell lines which are useful for the study of the early stage osteocytes; however, they are less useful for studying mature osteocyte signaling as they express low levels of sclerostin and FGF2313, both of which are mature osteocyte markers. Other cell lines including IDG-SW314 and Ocy45415, express high levels of sclerostin and FGF23 and are useful in studying the late osteocyte stage. Cell lines prove to be useful research tools; nevertheless, they do not come without limitations as they do not fully represent the biology of the primary cell. Different cell lines represent different developmental stages of the osteocyte maturity spectrum, and cell lines fail to represent the heterogeneity of primary osteocytes16,17.
To obtain pure cultures of primary osteocytes, researchers took advantage of the cre mouse model in which the 8-kb dmp1 promoter is used to drive green fluorescent protein (GFP) expression in osteocytes18,19. Dual transgenic mice (pOB-Col 2.3- GFP-cyan and DMP1-GFP-topaz) by Paic et al.19 and dmp1-topaz transgenic mice by Nakashima et al.20 have been used to obtain osteocyte populations. In which they employed sequential digestion and FACS of osteocytes expressing GFP to acquire cultures of primary osteocytes19,20. The direction of cre in the 10-kb dmp1 reporter mouse Ai9, which activates the tdTomato protein, was shown to be present in osteocytes, osteoblasts, muscles, and cells within the bone marrow. The 8-kb dmp1 promoter had the same expression pattern, but only a proportion of osteoblasts and bone marrow cells expressed the protein, which indicates that the 8-kb dmp1 promoter is more specific21. Despite this, results obtained using the 8-kb dmp1 promoter should be interpreted with caution, and gene expression profiles should be routinely carried out using osteocyte vs. osteoblast-specific markers to ensure that the population obtained is of high enough purity.
Osteoclast markers OSCAR and Dcstamp were found in hematopoietic non-depleted vs. depleted osteocyte populations, this finding led the authors to conclude that digests obtained from fractionation of 8-kb dmp1-topaz neonatal calvaria and GFP sorting are contaminated with hematopoietic cells. Contamination with hematopoietic cells could have been mitigated by tightening the GFP sort gate since GFP-positive hematopoietic cells had a reasonably lower GFP intensity than GFP-positive mesenchymal cells (osteocytes)22.
The methods for studying osteocytes in vitro have contributed to the recent wealth of information on osteocyte biology. However, osteocyte isolation remains a labor-intensive and lengthy procedure with low cell yields. The described method of bone digestion using collagenase and EDTA often up to fraction 823, takes several hours in which osteocyte viability is taxed. Researchers have reported using fractions (2&#8210;5) for cell sorting20, and have shown that the expression profile of genes associated with osteocytes versus those of osteoblasts confirms the success of isolating pure osteocyte populations20. In this article, we describe the process of obtaining fractions (2&#8210;5), and we compare the yield of osteocytes from each fraction starting with fraction 1 through 8 to determine the return of osteocytes in each fraction. We also describe the dissection of newborn dmp1-topaz mouse calvaria and calvarial digestion using collagenase and ethylenediaminetetraacetic acid (EDTA), as well as, preparation of cells for FACS.

<table><tbody><tr><td>BD FACSDiva software</td><td>BD Biosciences</td><td></td><td>Data aquisition and analysis</td></tr><tr><td>BD Falcon Tube</td><td>BD Biosciences</td><td>352235</td><td>12 x 75 mm Tube with Cell Strainer Cap, 35 &amp;mu;m nylon mesh.</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich, MO, USA</td><td></td><td></td></tr><tr><td>Collagenase</td><td>Wako, Osaka, Japan</td><td>034-22363</td><td>0.2% (w/v), crude collagenase mix sourced from C. histolyticum.</td></tr><tr><td>EDTA</td><td>Dojindo, Kumamoto, Japan</td><td></td><td>5mM EDTA prepared with 0.1% BSA</td></tr><tr><td>FACSAriaTM II</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Biowest, Nuaill&amp;eacute;, France</td><td></td><td></td></tr><tr><td>Isolation buffer</td><td></td><td></td><td>70mM NaCl, 10mM NaHCO, 60mM sorbitol, 3mM K2HPO4, 1mM CaCl2, 0.1% (w/v) BSA, 0.5% (w/v) glucose and 25 mM HEPES</td></tr><tr><td>Millex Sterile Filter Unit</td><td>Merck Millipore, Ireland</td><td>SLGV033RS</td><td>0.22&amp;mu;m</td></tr><tr><td>Nylon cell strainer</td><td>FALCON, NY, USA</td><td></td><td>40&amp;mu;m</td></tr><tr><td>Trypsin-EDTA</td><td>Life Technologies, NY, USA</td><td></td><td>0.5% x10. diluted to x1 in PBS</td></tr><tr><td>&amp;alpha;-MEM</td><td>Wako, Osaka, Japan</td><td></td><td>Containing 10% fetal bovine serum, 100 IU/ml penicillin G, and 100 &amp;mu;g/ml streptomycin</td></tr></tbody></table>

obtaining primary osteocytes through murine calvarial fractionation of gfp-expressing osteocytes

The osteocyte, once thought to be a passive resident of the bone given the backstage function of sensing mechanical loading, is now brought to the spotlight and has been shown to have multiple major functions like actively modifying the extracellular matrix and forming an endocrine organ with the lacunocanalicular system that encloses it sending messages to distant sites. Owing to the methods that made it possible to test the osteocyte in vitro from isolating primary osteocytes to osteocyte-like cell lines, osteocytes are now experiencing a resounding interest and a surge of knowledge on structure and function. Many aspects of the osteocyte biology and interaction with other molecular components are yet to be discovered. In this protocol, we describe in detail the efficient isolation of primary osteocytes from dmp1-topaz neonatal mouse calvaria, which express the green fluorescent protein in osteocytes, through cell fractionation and subsequently acquiring cultures of primary osteocytes by FACS.

The purpose of this protocol is to demonstrate the process of obtaining cultures of primary osteocytes from dmp1-topaz neonatal mouse calvaria through a fractionation process using collagenase to degrade the collagen matrix and EDTA for calcium chelation, after which cells are prepared for FACS to separate osteocytes from other cell populations.
Methods for obtaining primary osteocytes from neonatal mouse calvaria often describe the use of fractions (1&#8210;8) for sorting23. To test the efficiency of this method, we compared the yield of osteocytes obtained from one dmp1-topaz mouse calvaria, starting from fractions 1 through 8. Fractions 1&#8210;8 were sorted separately to determine the percentage and yield of osteocytes from each fraction. After the sort, we cultured osteocytes for 24 h on a 96-well plate to compare the density of the seeded cells. The density of osteocytes in fractions 2&#8210;5 is shown to be higher than that of fraction 1, and osteocyte density starts to decrease remarkably in fractions 6, 7, and 8 (Figure 1A).
Although the percentage of osteocyte obtained among all fractions is not statistically significant (Figure 1B), the density of osteocytes differs dramatically. Researchers20 have reported the use of fractions 2&#8210;5 for isolating osteocytes via FACS, and we show in Figure 1 that using fractions 2&#8210;5 optimizes the process for obtaining osteocytes and decreases the time of the sort.
Figure 2 shows the number of cells and gating strategy practiced to isolate GFP-positive from GFP-negative cells in which cells obtained through fractionation of GFP-negative mouse calvaria were used as control. Osteocytes obtained through this method were analyzed for gene expression of Dmp1 and SOST, which are known osteocytes markers. Dmp1 and SOST mRNA expressions are higher in osteocytes when compared to pre-sort fraction 2, which is known as a high osteoblast fraction (Figure 3A). Figure 3B shows the morphology of a GFP-positive osteocyte retaining a stellate shape with dendrites extending from the cell body cultured for 24 h on a plastic culture dish post sort.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61513/61513fig01.jpg" /> 
Figure 1: Efficiency of osteocyte fractionation.&#160;(A) Microscopic images of the density of osteocytes from fractions 1&#8210;8 seeded on a 96-well plate captured after 24 h of the sort. Fractions 2&#8210;5 have a higher cell density than fractions 1, and 6&#8210;8. Scale bar = 100 &#181;m. (B) Percentage of osteocytes obtained from fractions 1&#8210;8 as measured by the flow cytometer software. Results are derived from three separate representative experiments. Data are presented as a mean &#177; standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61513/61513fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61513/61513fig02.jpg" /> 
Figure 2: Isolation of GFP-positive osteocytes via fluorescence activated cell sorting.&#160;The upper panel represents cells isolated from GFP-negative C57Bl/6J littermate calvaria as GFP threshold control. The lower panel represents the number of cells and gating control practiced to isolate GFP-positive osteocytes from GFP-negative cells in fraction 2 obtained from dmp1-topaz mouse calvaria. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61513/61513fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61513/61513fig03.jpg" /> 
Figure 3: Osteocyte characterization post sort.&#160;(A) Relative mRNA expression of Dmp1 and SOST of fraction 2 cells cultured for 24 h and osteocytes cultured for 24 h post sort. Data are presented as a mean &#177; standard deviation. Statistical differences were detected by using Student&#8217;s t-test: * p &#60; 0.05, **p &#60; 0.01. (B) Microscopic image of an osteocyte retaining dendrites extending outwards from the cell body cultured for 24 h post sort. Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61513/61513fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes

This protocol describes the dissection of neonatal dmp1-topaz mouse calvaria and isolation of osteocytes expressing the green fluorescent protein through cell digestion and fractionation, in addition to osteocyte preparation for fluorescence activated cell sorting (FACS).

The osteocyte, once thought to be a passive resident of the bone given the backstage function of sensing mechanical loading, is now brought to the ...

obtaining-primary-osteocytes-through-murine-calvarial-fractionation

Nanyang Technological University

Research

JoVE Journal

Biology

2.1K Views.  Tohoku University Graduate School of Dentistry. Dit protocol beschrijft de dissectie van neonatale dmp1-topaas muizen calvaria en isolatie van osteocyten die het groene fluorescerende eiwit tot expressie brengen door celvertering en fractionering, naast osteocytenpreparaat voor fluorescentie geactiveerde celsortering (FACS).

Video: Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Developmental Biology

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

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

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, ...

Bioengineering

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

3D Modeling of Osteocytes for CFD Analysis to Study Mechanosensation in Bone

Analysis and Imaging of Osteocytes

Osteocytes are the bone cells that are thought to respond to mechanical strains and fluid flow shear stress (FFSS) by activating various biological pathways in a process known as mechanotransduction. Confocal image-derived models of osteocyte networks are a valuable tool for conducting Computational Fluid Dynamics&#160;(CFD) analysis to evaluate shear stresses on the osteocyte membrane, which cannot be determined by direct measurement. Computational modeling using these high-resolution images of the microstructural architecture of bone was used to numerically simulate the mechanical loading exerted on bone and understand the load-induced stimulation of osteocytes.
This study elaborates on the methods to develop 3D single osteocyte models using confocal microscope images of the Lacunar-Canalicular Network&#160;(LCN) to perform CFD analysis utilizing various computational modeling software. Prior to confocal microscopy, the mouse bones are sectioned and stained with Fluorescein isothiocyanate (FITC) dye to label the LCN. At 100x resolution, Z-stack images are collected using a confocal microscope and imported into MIMICS software (3D image-based processing software) to construct a surface model of the LCN and osteocyte-dendritic processes.
These surfaces are then subtracted using a Boolean operation in 3-Matic software (3D data optimization software) to model the lacunar fluidic space around the osteocyte cell body and canalicular space around the dendrites containing lacunocanalicular fluid. 3D volumetric fluid geometry is imported into ANSYS software (simulation software) for CFD analysis. ANSYS CFX (CFD software) is used to apply physiological loading on the bone as fluid pressure, and the wall shear stresses on the osteocytes and dendritic processes are determined. The morphology of the LCN affects the shear stress values sensed by the osteocyte cell membrane and cell processes. Therefore, the details of how confocal image-based models are developed can be valuable in understanding osteocyte mechanosensation and can lay the groundwork for future studies in this area.

This study outlines the method to visualize and develop three-dimensional (3D) models of osteocytes within the lacunar-canalicular network (LCN) for computational fluid dynamics (CFD) analysis. The generated models using this method help to understand osteocyte mechanosensation in healthy or diseased bones.

Osteocytes are the bone cells that are thought to respond to mechanical strains and fluid flow shear stress (FFSS) by activating various biological ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Intravital Longitudinal Imaging of Vascular Dynamics in the Calvarial Bone Marrow

Intravital longitudinal fluorescence microscopy imaging has emerged as a crucial technique for studying dynamic biological processes, notably in the context of tissue regeneration, tumor development, and therapeutic responses. Particularly, the calvarial bone marrow is a highly dynamic tissue, where the hematopoietic fate is interconnected with the surrounding microenvironment, with specialized vessels responding to normal and pathologic hematopoiesis. Traditional imaging of fixed tissues offers static information, often limiting a comprehensive understanding of these processes. 
The integration of transgenic animals expressing cell-specific markers, live cell tracers, advancements in imaging equipment, and the use of specialized chambers has elevated intravital microscopy to a pivotal tool for gaining insights into dynamic biological phenomena. One application of intravital imaging is the investigation of tumor vessel behavior and therapeutic effects. A newly designed 3D-printed titanium head fixation implant can be stably connected to the mouse skull and is suitable for longitudinal imaging during multiple sessions. 
The proposed protocol allows for the spatial and temporal examination of vascular dynamics in the calvarial bone marrow, including visualization and quantification of vascular heterogeneity, interaction with stromal and hematopoietic cells, and measurement of vascular functional parameters. Additionally, the technique enables the visualization of established vascular beds and the monitoring of therapeutic effects, stem cell mobilization, and the localization of chemotherapeutic compounds over time using two-photon microscopy. Overall, this intravital longitudinal imaging protocol provides a comprehensive platform for investigating both tumor vessel behavior and hematopoietic cell dynamics, offering valuable insights into the intricate processes governing these biological phenomena.

Intravital microscopy allows the study of dynamic biological processes such as tissue regeneration and tumor development. The calvarial bone marrow, a highly dynamic tissue, offers insights into hematopoiesis and vascular function. Using a biocompatible 3D-printed head fixation implant allows for repetitive longitudinal imaging, enhancing our understanding of tissue dynamics and the tumor microenvironment.

Intravital longitudinal fluorescence microscopy imaging has emerged as a crucial technique for studying dynamic biological processes, notably in the ...

Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes

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