We thank C. Park for reading the manuscript. This work was supported by the NIAMS under Award Number K01AR061434 and a Leukemia &#38; Lymphoma Society Fellowship Award (5127-09) to D.P and grants of the National Institutes of Health to C.P.L. and D.T.S. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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 Laborat...

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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...

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 diseases2,3. 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 repair5,6. 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 techniques8-10. 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+&#160;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.

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			<td height="21" style="height:21px;width:216px;">Extra fine Forceps</td>
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			<td style="width:140px;">Ethicon</td>
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			<td height="42" style="height:42px;width:216px;">Qtracker 705 non-targeted quantum dot</td>
			<td style="width:140px;">Invitrogen</td>
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			<td height="21" style="height:21px;width:216px;">Methocel 2%</td>
			<td style="width:140px;">OmmiVision</td>
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			<td height="42" style="height:42px;width:216px;">pIpC (Polyinosinic-polycytidylic acid)&#160;&#160;</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:195px;">P0913-50MG</td>
			<td>100 &#956;l (2.5 mg/ml in PBS) for 10 g of mouse</td>
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			<td height="42" style="height:42px;width:216px;">Mai Tai Tunable Ultrafast Lasers</td>
			<td style="width:140px;">Spectra Physics</td>
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			<td height="42" style="height:42px;width:216px;">Dual Calypso 491 + 532 nm DPSS laser</td>
			<td style="width:140px;">Cobolt AB</td>
			<td style="width:195px;"></td>
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			<td height="21" style="height:21px;width:216px;">Radius-635 HeNe laser</td>
			<td style="width:140px;">Coherent</td>
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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.

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...

Watch this Scientific Journal Video about Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair at JoVE.com

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.

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 ...

sequential-vivo-imaging-osteogenic-stemprogenitor-cells-during

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10.4K visualizações.  Harvard Stem Cell Institute.  A medida quantitativa da função progenitor óssea na consolidação da fratura requer alta resolução tecnologia de imagens em série. Aqui, os protocolos são fornecidos para o uso de microscopia intravital e rastreamento osteo-linhagem sequencialmente imagem e quantificar a migração, proliferação e diferenciação de células-tronco / progenitoras osteogênicas endógenos no processo de reparação de fratura óssea. 