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Developmental Biology

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

doi: 10.3791/61162 Published: September 16, 2020
Laura Ortinau1,2, Kevin Lei1, Youngjae Jeong1, Dongsu Park1,2,3


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.  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.


Fracture repair is a dynamic, multicellular process that is distinct from embryonic skeletal development and remodeling. During this process, the induction of injury signals from damaged tissue is followed by the rapid recruitment, proliferation, and subsequent differentiation of skeletal stem/progenitor cells, which are all critical for the overall stability and fixation of fractures1. In particular, early stages of fracture healing require soft callus formation, which is mainly attributed to periosteal resident cells2. When bones are injured, a subset of periosteal cells rapidly respond and contribute to newly differentiated cartilage intermediates and osteoblasts within the callus3, implicating the presence of a distinct skeletal stem/progenitor cell population within the periosteum. Therefore, the identification and functional characterization of the periosteum-resident skeletal stem cells (SSCs) constitute a promising therapeutic approach for degenerative bone diseases and bone defects4

SSCs are thought to reside in multiple tissue locations, including the bone marrow. Similar to bone marrow, osteogenic/chondrogenic SSCs have also been identified in the periosteum4,5,6.  These periosteal SSCs (P-SSCs) can be labeled with early mesenchymal lineage markers (i.e., Prx1-Cre5, Ctsk-Cre7, and Axin2-CreER8) during fetal bone development4,7,8,9,10. A significant limitation of these single genetic lineage tracing models is that there is substantial heterogeneity within labeled cell populations. Furthermore, they cannot distinguish labeled SSCs from their progeny in vivo. To address this limitation, we recently developed a dual reporter mouse (Mx1-Cre+Rosa26-Tomato+αSMA-GFP+) to distinctly visualize P-SSCs from bone marrow SSCs (BM-SSCs)11. With this model, it has been determined that P-SSCs are marked by Mx1+αSMA+ dual labeling, whereas more differentiated Mx1+ cells reside in the bone marrow, endosteal and periosteal surfaces, and cortical and trabecular bones11,12

Multiple cytokines and growth factors are known to regulate bone remodeling and repair and have been tested for the enhancement of skeletal repair in critical segment defects1,13. However, due to the cellular complexity of injury models generated through disruption of the bone compartment's physical barrier, the direct effects of these molecules on endogenous P-SSC migration and activation during healing are unclear. The functional characteristics and migratory dynamics of SSCs are often assessed in vitro by performing a transwell or scratch assay, in combination with cytokines or growth factors known to induce migration in other cell populations. Thus, interpretation of results from these in vitro experiments for application in their corresponding in vivo systems is challenging. Currently, in vivo assessment of skeletal stem/progenitor cell migration is not typically observed in real-time; rather, it is measured at fixed timepoints post-fracture5,7,14,15,16.  

A limitation of this method is that migration is not assessed at a single-cell level; rather, it is measured via changes in cell populations. Due to recent advances in live animal intravital microscopy and generation of additional reporter mice, in vivo tracking of individual cells is now possible. Using live animal intravital microscopy, we observed the distinct migration of P-SSCs from the calvaria suture niche to a bone injury within 24–48 h post-injury in Mx1/Tomato/αSMA-GFP mice. 

CCL5/CCR5 was recently identified as a regulatory mechanism influencing the recruitment and activation P-SSCs during the early injury response. Interestingly, there was no detection of substantial P-SSC migration in response to an injury in real-time. However, treatment of an injury with CCL5 produces a robust, directionally distinct migration of P-SSCs, which can be captured in real-time. Therefore, the objective of this protocol is to provide a detailed methodology for recording the in vivo migration of P-SSCs in real-time after treatment with CCL5.

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All mice were maintained in pathogen-free conditions, and all procedures were approved by Baylor College of Medicine’s Institutional Animal Care and Use Committee (IACUC).

1. Mouse preparation

  1. Cross Mx1-Cre17 and Rosa26-loxP-stop-loxP-tdTomato (Tom)18 mice (purchased from the Jackson Laboratory) with αSMA-GFP mice (provided here by Drs. Ivo Kalajzic and Henry Kronenberg) to generate Mx1-Cre+Rosa26-Tomato+αSMA-GFP+ (Mx1/Tomato/αSMA-GFP) reporter mice (Figure 2A). 
  2. For Mx1-Cre induction, inject mice with 25 mg/kg pIpC every other day for 10 days. 
  3. Irradiate mice with 9.5 Gy 1 day prior to intravenous transplantation of 1 x 106 whole bone marrow mononuclear cells from wild-type C57BL/6 mice (WT-BMT)19.  
  4. After 6 to 8 weeks of recovery, subject mice to the in vivo imaging of periosteal cell migration protocol described below.
    NOTE: Irradiation and WT-BMT of Mx1-Cre mice are necessary to eliminate endogenously Mx1-labeled hematopoietic cells that may respond to an injury. Six to eight weeks post-irradiation, the host's hematopoietic cell count is at <5% and thus ready for imaging19.

2. Mouse restraint and imaging mount

  1. Create a mouse restraint with a 50 mL conical tube (Figure 1A).
    1. Using pointed scissors, poke a hole through the bottom of the conical. Cut from the bottom hole to the top of the conical (the cut side will be the top of the restraint). 
    2. Make a cut approximately 4–5 cm long parallel to the previous cut and halfway down (~2 cm) from the top of the restraint. Remove this section of the conical and smooth the edges were necessary.
  2. Construct a conical tube imaging mount using an adjustable angle plate, a 0.5” optical post, and a right-angle clamp for the 0.5” post, 0.1875” Hex (Figure 1B and Table of Materials).
    1. Adjust the angle plate to a 35° angle. On the left side of the angle plate, screw the optical post into the second hole from the back of the plate.
    2. On the right side of the adjustable plate, insert a spring-loaded 0.1875” Hex-locking 0.25” thumbscrew into the second hole from the front and last holes on the plate. The right-angle clamp will be used with the optical post to secure the conical mouse restraint in place.

3. Preoperative surgical procedure

  1. Mouse anesthesia
    1. For pain management, subcutaneously inject the mouse with sustained release buprenorphine (1 mg/kg BW) 30–60 min prior to surgery.
    2. Anesthetize mice using rodent III CCM combination (combo III) anesthesia containing 37.5 mg/mL ketamine, 1.9 mL/mg xylazine, and 0.37 mg/mL acepromazine. The appropriate dosage for mice is 0.75–1.50 mL per 1 kg of body weight. The dose will last 30–45 min.
    3. Once the mouse is anesthetized, apply ophthalmic ointment to the eyes with a sterile cotton swab to prevent eye dehydration.
    4. For long-term imaging (>45 min), administer anesthesia again to maintain proper anesthetic depth as follows.
      1. Prepare a 1 mL syringe with a sufficient amount of combo III to maintain anesthesia for the planned imaging duration (0.75 mL/kg BW per 30 min of additional sedation). Attach a 25 G butterfly needle infusion set with 12” tubing to the syringe and push anesthetic through the tubing and needle.
      2. Insert the butterfly needle intraperitoneally, then tape the needle in place. 
        NOTE: This is how the additional combo III will be administered during imaging such that there is no need to move the animal or disrupt the plane of view.
  2. Mouse restraint and preparation of the surgical site
    1. Use clippers to remove as much hair as possible from the top of the head. Apply depilatory cream to the shaved area to remove any remaining hair at the surgical site. 
    2. Ensure that the mouse is fully anesthetized by a lack of response to toe pinches. After 15 min, if the mouse is not fully anesthetized, administer a second smaller dose of combo III (<0.75 mL/kg BW).
    3. Place the mouse in the restraint tail-first, sliding it far enough backwards such that the nose slightly hangs over the edge of the conical.  
    4. Tape the front paws to the bottom of the conical tube using medical tape.
    5. Place conical tube with mouse on an imaging mount that is set at a 35° angle and secure using the right-angle clamp (Figure 1C).
    6. Once the mouse is secure in the imaging mount, clean the surgical site 3x with alternating scrubs of betadine scrub and 70% isopropyl alcohol. Again, check that the animal is fully sedated.
    7. Move the mouse to a clean absorbent pad and place a drape over the mouse to create a large sterile field.   

4. Surgical procedure

  1. Opening incision
    1. Using fine-tipped forceps, pick up the skin immediately medial to the right ear. With microdissection scissors, cut the skin being held by the forceps. 
      NOTE: Initiating the incision using the method described above creates a rounded incision that is more suitable for suturing after the procedure.
    2. Make a transverse incision starting from the initial incision toward the left ear (<1 cm).  Make another incision, starting from the initial incision, towards the nose of the animal (~2–3 mm past the right eye).
    3. Separate the skin from the periosteum using the forceps and scissors to gently cut through the connective tissue separating the skin from calvaria periosteum. 
    4. Using a sterile cotton swab, apply some ophthalmic ointment to the top of the skin flap.  Gently pick up the skin flap and fold it over the left eye. The intersection of the sagittal and coronal sutures should be clearly exposed (Figure 1E).
      NOTE: The ophthalmic ointment is used to keep the skin flap in place.
    5. Flush the open surface with sterile PBS to clean the area of any blood and residual hair. 
      NOTE: If necessary, use fine-tipped forceps to remove hairs that remain after flushing, but be careful not to damage the periosteum.
  2. Microfracture
    1. To generate microfractures on the calvaria, remove the plunger of a 29 G insulin syringe and cut the back end of the syringe, around the 300–400 uL marks.
      NOTE: This step makes it easier to maneuver under a dissection scope when producing the injury.
    2. Place the tip of a bevel (one to two needle widths) towards the nose from the coronal suture and one to two needle widths to the right of the sagittal suture (Figure 1E).
    3. Gently rotate the syringe clockwise, then counterclockwise several times.
      NOTE: In young mice, a very small amount of downward pressure is needed to create a circular microfracture. Be careful not to let the needle penetrate the brain, as this will cause bleeding and interfere with imaging.
    4. Use a 27 G needle to widen the microfracture to ~0.4 mm. If bone fragments remain in the microfracture, flush the area with PBS. If bone fragments still remain in the microfracture, use the 29 G needle to gently scoop them out. 
    5. Repeat steps 4.2.1–4.2.4 to create an injury on the left side of the sagittal suture.
      NOTE: At this point, there are two options: 1) immediately proceed to CCL5 treatment (section 4.3) and imaging (section 5); or 2) close the surgical site following the postoperative procedures in section 6. At 24 h later, anesthetize the mouse again, reopen the surgical sight, treat the injury with CCL5 (section 4.3), and perform intravital imaging (section 5) and postoperative care (section 6).
  3. CCL5 treatment
    1. From a stock of RANTES (100 ng/mL), make a solution of 10 ng/mL using basement membrane matrix (Table of Materials). Keep this solution on ice to keep the matrix in liquid form.
    2. Treat each injury with 2 μL of CCL5 (10 ng/μL) by using a 2-20 μL pipette (this pipette tip has a larger diameter, which is more effective when using a viscous liquid). Allow the matrix to solidify.
    3. Once the matrix has solidified, gently cover the calvaria with the skin flap to ensure the tissue does not become dehydrated. Incubate for 1 h prior to imaging.

5. Intravital imaging

  1. Microscope settings
    1. Turn on the multiphoton laser (femtosecond titanium-sapphire laser). Set the wavelength to 880 nm and adjust the power for second harmonic generation (SHG) imaging (440 nm) of the bone.
    2. Turn on the 488 nm and 561 nm solid state lasers for excitation of GFP- and tdTomato-expressing cells, respectively.
    3. Turn on PMT (photomultiplier tube) detectors for second harmonic generation (405–455 nm detection) and GFP (505–550 nm detection) signals. Turn on HyD 3 detector for tomato (590–620 nm detection) signal. 
  2. Mounting
    1. Before moving the mouse to the scope, check the visual plane of the calvaria by gently pressing the back of the mouse’s head. If the plane is not level, adjust the position by gently rotating the mouse restraint to the left or the right. 
      NOTE: This is a critical step to ensure sufficient image quality.
    2. Apply sterile 2% methylcellulose in water (w/v) to cover the entire calvaria and skin flap and prevent drying of the tissue.
    3. Place the mouse on the XYZ-axis motorized microscope stage (Figure 1G). Using the epifluorescent light, align the mouse so that 1) it is facing to the microscope and 2) the intersection of the coronal and sagittal sutures is the centered reference point of the stage.
    4. Place the glass cover on the imaging area and double-check the alignment (Figure 1H).
  3. Time-lapse imaging
    1. Use a low magnification lens (25x water immersion objective with NA = 0.95) to scan the calvaria. Acquire a reference image of the sagittal and coronal suture intersection.
    2. Using the SHG and fluorescent signals from the cells, locate each injury sight and record the XYZ coordinates as well as the distances from the sagittal and coronal sutures (Figure 2B).
    3. Pick a location on either the coronal or sagittal sutures for long-term imaging. Take a detailed Z-stack image of this area from reference during imaging. 
      NOTE: Since the sutures represent a known P-SSC niche, this is where the cells will be migrating from in response to the calvaria injury.
    4. Use the time-lapse software settings to record an image every 1 min for at least 1 h. In the time between snapshots, compare the current field of view with the initial field of view. If they are different, use the Z-stack image to determine in which direction the field of view needs to be adjusted.

6. Postoperative animal care

  1. After imaging, rinse the exposed calvaria with sterile PBS to remove the methylcellulose. 
  2. To close the incision, gently place the skin flap over the calvaria. Use sterile cotton swabs to remove residual ophthalmic ointment and dry the incision site.
  3. Using monofilament nylon 5-0 size nonabsorbable sutures with an attached C-1 reverse cutting needle, close the surgical site. Use a sterile cotton swab to apply triple antibiotic ointment to the closed incision.
    NOTE: Scarring is reduced with high quality suturing techniques and minimum bleeding.
  4. Place mouse in a clean cage on a warm surface and check every 15 min until sternal recumbency is achieved.
  5. Administer a second dose of sustained release buprenorphine 72 h post-surgery.
  6. Monitor daily for huddling behavior, ruffled fur, and/or lack of ambulation until sutures are removed (~1 week). 

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Representative Results

Skeletal progenitors have been proposed to have migratory or circulatory potential20. Recently, Mx1-Cre+Rosa26-Tomato+αSMA-GFP+ (Mx1/Tomato/αSMA-GFP) reporter mice were generated, in which P-SSCs are marked by Mx1+αSMA+ dual labeling (Figure 2A,B). Substantial migration of Mx1+αSMA+ P-SSCs occurred out of the suture mesenchyme within 24–48 h post-injury11.  Also tested was the possibility of detecting in vivo P-SSC migration in real-time 24 h after a calvaria defect. Little to no migration of Mx1+αSMA+ P-SSCs was observed within 1 h of imaging (Figure 2C).

Mx1+αSMA+ P-SSCs uniquely express the CCL5 receptor known as CCR511. Thus, treatment of a calvaria defect 24 h post-injury with CCL5 induced directionally distinct migration away from the coronal suture and towards the injury sight (Figure 2D,E). Within 1 h of imaging, one of the Mx1+αSMA+ P-SSCs migrated approximately 50 μm toward the injury (Figure 2E). Other Mx1+αSMA+ P-SSCs were also observed migrating in response to CCL5 treatment but to a lesser extent. 

Figure 1
Figure 1: Set-up for in vivo imaging of mouse calvaria. Representative images of the mouse restraint (A) and imaging mount (B) used during live imaging. (C) Mouse in the restraint and secured to the imaging mount. (D) Schematic representation of mouse calvaria structure, identifying the frontal (FS), coronal (CS), sagittal (SS), and lambdoid (LS) sutures. (E) Representative placement of calvaria microfracture injuries. (F) Postoperative surgical suture placement for optimal healing and future imaging. Representative images of mount placement on microscope stage (G) and coverslip placement (H). Please click here to view a larger version of this figure.

Figure 2
Figure 2: Real-time in vivo imaging of Mx1+αSMA+ P-SSC migration. (A) Schematic of Mx1/Tomato/αSMA-GFP dual reporter mice preparation.  Mice were injected intraperitoneally with 25 mg/kg pIpC every other day for 10 days (1). Mx1/Tomato/αSMA-GFP mice were lethally irradiated with 9.5 Gy (2) 1 day before intravenous transplantation of 1 x 106 whole bone marrow mononuclear cells from WT C57BL/6 mice (3).  After 6–8 weeks of recovery (when the host's hematopoietic cells wer less than 5%), mice were subjected to bone injury experiments. Mx1+ (Tomato+), αSMA+ (GFP+), Mx1+αSMA+ (Tomato+GFP+), and bone (blue). (B) Maximum Z-projection of a representative calvaria injury (white circle) and imaging (white square) locations in relation to sagittal (SS) and coronal (CS) sutures of Mx1/Tomato/αSMA-GFP mouse.  Dotted lines represent calvaria sutures. (C) In vivo imaging of Mx1+αSMA+ P-SSCs response 24 hours post-injury. Scale bar = 25 μm. (D) 24 h post-injury, CCL5 was administered at the injury sight, and in vivo imaging was performed 1 h later in relation to the coronal suture (CS). White square represents location of cell migration images shown in (E). Scale bar = 75 μm.  (E) Migration of Mx1+αSMA+ P-SSCs on the bone (blue) surface toward the injury sight (top right). Scale bar = 25 μm. In (C,D,E), the numbers indicate the time of imaging. Yellow arrow indicates the direction of injury location. Asterisk indicates the migratory path of Mx1+αSMA+ P-SSC. The image is representative of results from three to five mice. Please click here to view a larger version of this figure.

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During bone healing, periosteal cells are the main source of newly differentiated chondrocytes and osteoblasts within an injury callus3. Similar to bone marrow, osteogenic/chondrogenic SSCs have also been identified in the periosteum4,5,6. Assessing endogenous P-SSC functional characteristics is technically challenging. Often, migratory dynamics of SSCs are assessed in vitro, making interpretation of their corresponding in vivo systems challenging. Due to recent advances in live animal intravital microscopy and generation of additional reporter mice (Mx1/Tomato/αSMA-GFP), in vivo tracking of individual cells is now possible. Thus, this method allows the in vivo recording of CCL5 induced P-SSCs migration in real-time.

There are several technical challenges associated with this method that can influence imaging consistency. A major technical challenge with this method is maintaining the Z-axis plane of view. Z-axis drift (Z-drift) is a common issue in long-term imaging of fixed and live samples, and it is largely attributed to temperature changes in the imaging environment.  Although this artifact cannot be completely counteracted, covering the stage, objective lenses, and supporting structures helps to reduce Z-drift21.

Furthermore, obtaining a Z-stack series of the imaging location prior to time-lapse imaging will establish a reference of the Z-axis that can be referred to between image acquisitions (i.e., images are typically taken every 30-60 s, which allows time for adjustments to the Z-axis, if necessary). Another technical challenge is making a consistent microfracture that is the same size, shape, and distance from the calvaria sutures. The distance from the coronal and sagittal sutures is critical, because the calvaria sutures are a niche for quiescent SSCs10. The best method to reduce variability of the microfracture is to practice this technique in mice that are not included in the final analysis. 

Despite the limitations, this method provides a unique system to assess individual cellular response in vivo to a bone injury. Since fracture repair is a highly complex processes involving multiple cell types, there are several aspects that are not fully understood. One of these includes the early migratory response of P-SSCs toward an injury. While CCR5/CCL5 is a unique mechanism by which P-SSCs are recruited to an injury, it is possible that additional cytokines and growth factors play an important role in fracture healing. Previously, the treatment of fractures with various cytokines and growth factors has resulted in highly variable outcomes in overall fracture healing22. This imaging method provides a unique platform to determine the physiological effects of potential drug/cytokine/growth factor therapies on individual cellular responses. Finally, it provides in vivo mechanistic data that can support a specific therapy’s enhancement of fracture healing.

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L.C.O. and D.P. disclose a pending patent entitled “Periosteal Skeletal Stem Cells in Bone Repair” (BAYM.P0264US.P1). The remaining authors declare no competing interests.


This work was supported by the Bone Disease Program of Texas Award, the Caroline Wiess Law Fund Award, and the NIAMS of the National Institutes of Health under award numbers R01 AR072018, R21 AG064345, R01 CA221946 to D.P.  We thank M.E. Dickinson and T.J. Vadakkan in the BCM Optical Imaging and Vital Microscopy Core and the BCM Advanced Technology Cores with funding from NIH (AI036211, CA125123, and DK056338).



Name Company Catalog Number Comments
½” optical post ThorLabs TR2 For imaging mount
1 mL syringe BD 309659
27G needle BD PercisionGlide 305111
29G insulin syringe McKesson 102-SN05C905P
50 mL conicol tube Falcon 352098 For mouse restraint
Adjustable angle plate Renishaw R-PCA-5023-50-20 For imaging mount
Alcohol wipes Coviden 6818
betadine surgical scrub Henry Schen 67618-151-16
Buprenorphine SR-LAB ZooPharm 1mg/mL Sustained Release
Combo III Obtained from staff veterinarian N/A 37.6 mg/mL Ketamine; 1.9 mg/mL Xylazine; 0.37 mg/mL Acepronazine
Coverslip Fisher 12-545-87 24 x 40 premium superslip
Fine tip forcepts FST 11254-20
Ketamine KetaVed 50989-161-06 100 mg/mL
Leica TCS SP8MP with DM6000CFS Leica Microsystems N/A
Matrigel R & D Systems 344500101
Medical tape McKesson 100199 3" x 10 yds (7.6 cm x 9.1 m)
Methocellulose Electron Microscopy Sciences 19560
Microdissection scissors FST 1456-12
Motorized stage Anaheim automation N/A
Needle holder FST 12500-12
Nonabsorbable sutures McKesson S913BX monofilament nylon 5-0 nonabsorbable sutures with attached C-1 reverse cutting needle
Opthalmic ointment Rugby 0536-1086-91
RANTES Biolegend 594202 10 µg/50 µL
Right-angle clamp for ½” post, 3/16” Hex ThorLabs RA90 For imaging mount
Spring-loaded 3/16” Hex-locking ¼” thumbscrew ThorLabs TS25H For imaging mount
Sterile cotton swabs Henry Schen 100-9249
Sterile DPBS (1x) Corning 21-030-CV
Sterile drapes McKessen 25-517
Surgical gloves McKessen 3158VA
Triple antibiotic ointment Taro Pharmaceuticals U.s.a., Inc. 51672-2120-2
Vacutainer blood collection set BD REF 367298 25G butterfly needle infusion set with 12" tubing



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Cite this Article

Ortinau, L., Lei, K., Jeong, Y., Park, D. Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice. J. Vis. Exp. (163), e61162, doi:10.3791/61162 (2020).More

Ortinau, L., Lei, K., Jeong, Y., Park, D. Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice. J. Vis. Exp. (163), e61162, doi:10.3791/61162 (2020).

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