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

Summary

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.

Abstract

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.

Introduction

Degenerative bone diseases and age-related bone loss leading to a high risk of osteoporotic fracture has become a major challenge in public health¹. Bone maintenance is controlled by bone-forming osteoblasts and bone-resorbing osteoclasts. Defects of bone forming cells are a main cause of age-related bone loss and degenerative bone diseases^2,3. While extensive research has focused on the improvement of fracture healing, the discovery of reliable drugs to cure degenerative bone diseases and to reverse the weakness of osteoporotic fractures remains an important issue. Thus, studying the source of bone forming cells and their control mechanisms in bone regeneration and repair provides a novel insight to enhance skeletal regeneration and reverse bone loss diseases.

The existence of multipotent mesenchymal cells in bone marrow has been proposed based on the identification of clonogenic populations that could differentiate into osteogenic, adipogenic and chondrogenic lineages ex vivo⁴. Recently, multiple studies have reported that skeletal/mesenchymal stem cells (SSCs/MSCs) are a natural source of osteoblasts and are critical for bone turnover, remodeling, and fracture repair^5,6. In addition, our lineage-tracing study revealed that mature osteoblasts have a unexpectedly short half-life (~60 days) and are continuously replenished by their stem/progenitor cells in both normal homeostatic and fracture repair conditions⁶. However, the in vivo identity of stem cells and how such cells react to fracture injury and supply bone-forming cells are unclear. Therefore, it is important to develop a method that is able to analyze the migration, proliferation and differentiation of endogenous SSCs/MSCs in under physiological circumstances.

Fracture repair is a multi-cellular and dynamic process regulated by an array of complex cytokines and growth factors⁷. The most popular approach for fracture studies is to use an animal model with long-bone fracture and to analyze bones by bone sectioning and immunofluorescent techniques^8-10. This repair process can be monitored by multiple imaging techniques including micro-CT¹¹, near-infrared fluorescence¹², and chemiluminescence imaging¹³. However, each technique has certain limitations and there has been no effective way to monitor SSCs/MSC function at the cellular level in vivo. Recently, confocal/two-photon intravital microscopy has been developed and used to detect transplanted cancer cells and hematopoietic stem cells in the context of their bone marrow microenvironment even at single-cell resolution in living animals¹⁴. By combining this technology with a series of lineage tracing models, we were able to define that osteogenic stem/progenitor cells can be genetically marked by transient activation of the myxovirus resistance-1 (Mx1) promoter and Mx1-induced progenitors can maintain the majority of mature osteoblasts over time but do not participate in the generation of chondrocytes in the adult mouse⁶. In addition, we demonstrated that Mx1-labeled OSPCs supply the majority of new osteoblasts in fracture healing⁶.

Here, using osteo-lineage tracking models and intravital microscopy, a protocol is provided to define the in vivo kinetics of Mx1⁺ osteogenic stem/progenitor cells in fracture repair. This protocol offers sequential imaging to track the relocation of osteogenic stem/progenitors into fracture sites and the quantitative measurement of osteoprogenitor expansion in the early repair process. This approach may be useful in multiple contexts including the evaluation of therapeutic candidates to improve bone repair.

Protocol

1. Mice and Preconditioning

Note: All mice were maintained in pathogen-free conditions and all protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the Massachusetts General Hospital. All surgery should be performed under sterile condition using autoclaved sterile equipment. Mx1-Cre¹⁵, Rosa26-loxP-stop-loxP-EYFP (Rosa-YFP), and Rosa26-loxP-stop-loxP-tdTomato (Rosa-Tomato), were purchased from Jackson Laboratories. Osteocalcin-GFP mice were provided by Dr. Henry Kronenberg. For the quantitative analysis of OSPC migration and proliferation in vivo, Mx1-Cre⁺Rosa-YFP⁺ (single color-reporter) mice were used. For more detailed tracking of their differentiation into Osteocalcin⁺ mature osteoblasts, we used trigenic Mx1-Cre⁺Rosa-Tomato⁺Ocn-GFP⁺ mice.

1. To label Mx1⁺ cells in vivo, prepare polyinosinic-polycytidylic acid (pIpC, 2.5 mg/ml) in sterile PBS solution and inject 200 μl for 20 g of Mx1-Cre⁺ reporter mouse intraperitoneally once every other day for 10 days.
2. To eliminate resident Mx1⁺ hematopoietic cells, irradiate mice with a single dose of 9.5 Gy. After 24 hr of irradiation, transplant wild type bone marrow cells (1x10⁶ cells/mouse) intravenously¹⁶. Note: Since Mx1-inducible cells include hematopoietic cells and hematopoietic-derived osteoclasts, the elimination of Mx1⁺ hematopoietic cells improves the imaging quality and quantitation of Mx1⁺ osteogenic cells.
3. After bone marrow transplantation, monitor animals for four to six weeks in order to achieve a successful repopulation of donor marrow cells.

2. Mouse Preparation

1. Anesthetize mouse by intraperitoneal injection of 50 μl of ketamine/xylazine (100 mg/kg, xylazine 12 mg/kg body weight) using an IACUC-approved procedure. Note: Duration of the effect may be extended by an additional dose of ketamine/xylazine.
2. Determine if the mouse is fully anesthetized by the lack of response to toe and/or tail pinches. (Optional) When the mouse is anesthetized, secure an isoflurane gas mask over the nose by taping. Adjust the level of isoflurane to ensure the animal is fully sedated.
3. Clip the scalp hair using an electric trimmer or small scissors. Remove the hair fragments and sterilize the exposed skin with 70% alcohol swab. Apply tear gel to prevent corneal dehydration.

3. Microfracture Injury

1. After ensuring the animal is fully sedated, make a single reverse L-shaped incision to open the scalp skin. Briefly, make a first transverse incision (less than 1 cm) starting from one ear to another ear. Turn ~60° and continue to incise toward the nose until ~2-3 mm away from nose. Note: This incision minimizes the formation of scar tissues above the area to be imaged.
2. Separate the skin flaps by pulling toward the sides with forceps. Both frontal bones and the intersection of sagittal and coronal sutures should be clearly exposed.
3. Clean the open surface by flushing with sterile PBS and gentle wiping with sterile gauze or cotton swabs until all the residual hairs are eliminated.
4. To generate microfractures on the calvarium, hold the mouse head with one hand and a 30 G needle with another hand. Make a micro-puncture (~0.2 mm diameter wound with < 1 mm depth) on the left frontal bone near the intersection of the sagittal and coronal sutures by inserting the tip of a 30 G needle with gentle pressure and twisting motion. Caution: It is critical to avoid the needle from penetrating into the brain, thereby minimizing bleeding and tissue damage.
5. Switch to a bigger needle (20 or 25 G) and widen the puncture hole by twisting the needle (~0.5 mm diameter).
6. Repeat step 3.3-3.5 to generate second puncture on the contralateral frontal bone.
7. Wipe out the injured spots by continuous dropping of PBS until the bleeding stops.
   Note: Bleeding on fracture sites will interfere with appropriate imaging and induce scar formation.
8. Apply a drop of sterile physiological saline solution or 2% Methocel on the skull to avoid drying. Note: Do not allow the skull surface to dry, which will reduce image clarity. It is important to use a sufficient amount of gel or saline to cover the area.

4. Intravital Imaging

1. (Optional) Before imaging, some mice are injected with a vascular dye to visualize the blood vessels. Inject retro-orbitally with 20 μl of non-targeted Qtracker 705 (2 μM solution in 50 mM borate buffer) diluted in 80 ml of PBS. Use an insulin syringe to minimize reagent waste. If the signal is not sufficiently bright, additional amounts might be required.
2. Turn on the scanner. Note: A polygon-based laser scanner allows simultaneous multi-channel image acquisition at video rate (30 frames per sec). Video rate scanning is very important for live animal imaging.
3. Turn on the multi-photon laser (femto-second titanium:sapphire laser). Set the wavelength to 880 nm and adjust the power for second harmonic generation imaging (440 nm) of the bone. For GFP and tdTomato excitation, turn on the 491 nm and 561 nm solid-state lasers.
4. Turn on the PMT (photomultiplier tube) detectors for each signal (a 435 ± 20 nm band-pass filter for second harmonic generation, a 528 ± 19 nm band-pass filter for GFP, and 590 ± 20 for tdTomato.) (Optional) Use a 638 nm helium–neon laser and a PMT with a 695 ± 27.5 nm band-pass filter to detect Qtracker 705 signal.
5. Place the animal on a XYZ-axis motorized microscope stage. Keep the mouse in the most comfortable position with an electric heating pad to help maintain body temperature (this reduces the risk of animal loss). Use tape to hold the mouse to minimize movement during imaging and to keep the imaged area as horizontal as possible.
6. Apply a drop of warm 2% methylcellulose gel or physiological saline solution on the skull to avoid drying. Put a cover glass on the imaging area. Use a low magnification lens (30x water-immersion objective with 0.9 NA) to scan the calvaria.
7. Using the XYZ-axis controller, find the surface of calvaria by detecting the SHG signal from bones and identify a crucial landmark location, such as the intersection between the sagittal sutures and coronal sutures. Acquire an image and record the XYZ coordinates.
8. Continue to search for the location of the injury by observing SHG and fluorescence signals.
9. When injury sites or the region of interest are found, acquire an image of the best focal plane containing SHG and fluorescence signals from the cells of interest. Save XYZ coordinates and the distance to the intersection of the sagittal and coronal sutures to define their precise location for the next rounds of imaging.
10. To collect 3D cellular and bone structures of fracture injury, record images by Z-stacks (2 - 5 μm interval) with ~100 μm depth from endosteal bone surface. After the completion of imaging of one side of injury, repeat imaging process for next injury sites.

5. Post Operation Procedures

1. After imaging, rinse imaging site with sterile saline solution to remove the methylcellulose gel from the skull. Using a sterile cotton swab, apply a small amount of triple antibiotic ointment on the surface. Cover the skin flaps and re-close the scalp by surgical suturing. This can be done with a hypoallergenic suturing thread (absorbable polyglactin suture) and 5-0 size suture needle. Caution: Good suturing technique minimizes scar formation.
2. Treat all animals with IP injection of 0.05-0.1 mg/kg buprenorphine every 12 hr for 48 hr following surgery. Keep animals in a warm recovery chamber until they regain sufficient consciousness. After 3 to 5 days, reopen the suture and repeat intravital imaging steps to track the cellular change during fracture healing.

Results

The stabilized long bone fracture model has been popular in fracture studies. However, long bone or large fracture models cause multiple tissue damage and therefore, have a limitation in quantitative measurement of bone cell function. We developed a minimally invasive injury (less than 1 mm diameter with minimal or no invasion into the dura mater) on calvarial frontal bones with needle drilling (Figures 1A-1C). We chose a top view of the calvarial frontal bones for in vivo live imaging of microf...

Discussion

The regulation of skeletal stem cells may be of great importance for defining better methods to achieve bone regeneration. Quantitative and sequential imaging at the cellular level has been technically challenging. Although the mouse long-bone fracture model has been widely used and suitable for biomechanical studies ¹⁷, its deep tissue location, uneven fracture size, soft tissue damage, and the application of stabilizing fixators have limited sequential intravital imaging. Here, a method to overcome these lim...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6J (H-2b)	Jackson Laboratories (Bar Harbor, ME)	000664
Ketamine Hydrochloride Injection	Bionichepharma	67457-001-10	Vial size: 10 ml (50 mg/ml)
Xylazine Sterile Solution	Lloyd Inc.	NADA# 139-236
Buprenorphine Hl	BEDFORD LAB	NDC 55390-100-10	Vial: 0.3 mg/ml, Doses: 0.05-0.1 mg/kg
DPBS, 1X	CORNING cellgro	21-031-CV
Alcohol Prep Pads (70% Isopropyl alcohol)	Kendall WEBCOL	5110
Fine Surgical Scissor	F.S.T	14568-09
Extra fine Forceps	F.S.T	11150-10
VICRYL*Plus Suture	Ethicon	VCP490G
Qtracker 705 non-targeted quantum dot	Invitrogen	Q21061
Methocel 2%	OmmiVision
pIpC (Polyinosinic-polycytidylic acid)	Sigma	P0913-50MG	100 μl (2.5 mg/ml in PBS) for 10 g of mouse
Mai Tai Tunable Ultrafast Lasers	Spectra Physics
Dual Calypso 491 + 532 nm DPSS laser	Cobolt AB
Radius-635 HeNe laser	Coherent