The murine bone marrow injury model is a useful tool for studying postnatal osteogenesis through intramembranous ossification. This simplified surgical protocol describes the bone marrow ablation procedure for downstream assessment of new bone formation and cellular responses following injury.
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Method Article
Improved Methodology for Studying Postnatal Osteogenesis via Intramembranous Ossification in a Murine Bone Marrow Injury Model
* These authors contributed equally
In This Article
Summary
Abstract
Long bone injuries heal through either endochondral or intramembranous bone formation pathways. Unlike the endochondral pathway that requires a cartilage template, the process of intramembranous ossification involves the direct conversion of skeletal stem and progenitor cells (SSPCs) into bone-forming osteoblasts. There are limited surgical methods to model this process in experimental mice. Here, we have improved upon a bone marrow injury model in mice to facilitate the study of bone repair via intramembranous ossification and to assess postnatal regulators of osteogenesis. This method is highly reproducible and user-friendly, and it allows temporal assessment of new bone formation in a short period (3-7 days post-injury) using micro-computed tomography (µCT) and frozen section histology. Furthermore, the contributions of SSPCs and mature osteoblasts can be readily assessed using a combination of fluorescent reporter mice and this intramembranous bone marrow injury model. In clinical contexts, intramembranous bone formation is relevant for healing critical size defects, stabilized fractures, cortical defects, trauma from tumor resections, and joint replacements.
Introduction
Long bone injuries heal through the endochondral or intramembranous bone formation pathway. Unlike the endochondral pathway, which requires a cartilage precursor step, bone repair via intramembranous ossification involves direct conversion of skeletal stem and progenitor cells (SSPCS) into bone-forming osteoblasts1. Clinically, this process is relevant for bone healing in a variety of contexts, including critical size defects, stabilized fractures, cortical defects, distraction osteogenesis, trauma from tumor resections, and osseointegration of joint replacement implants2,3. I....
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Protocol
The following procedures were performed with approval from the University of Connecticut Health Center Institutional Animal Care and Use Committee (IACUC). All surgeries were performed under sterile conditions as outlined by the National Institutes of Health (NIH) guidelines. Pain and risk of infections were managed with proper analgesics and antiseptics to ensure a successful outcome.
1. Preparation of surgical area and instruments
- Prepare the surgical procedure room by disinfecting working surfaces with a clinical-grade disinfectant cleaner.
NOTE: The surgeon should wear appropriate personal protective ....
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Results
The success of the mechanical bone marrow (BM) ablation procedure was validated by X-ray imaging, while the 26G needle was inserted into the medullary cavity (Figure 2A). When X-ray imaging indicated that the needle was inserted at an improper angle or penetrated through the bone, the needle was re-inserted at the proper angle and re-imaged to confirm accurate placement. When a successful position could not be attained, the animal was excluded from analysis. While radiographic two-dimensiona.......
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Discussion
The goal was to establish a reliable and reproducible method to study the molecular and cellular processes governing bone repair, focusing on the direct conversion of skeletal stem and progenitor cells (SSPCs) into bone-depositing osteoblasts in the bone marrow space/cavity. The mechanical bone marrow ablation method described here is an optimized and validated protocol for bone marrow ablation by mechanical injury, which allows for the investigation of intramembranous bone formation. Several steps in this protocol are c.......
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Disclosures
The authors have no disclosures to report.
Acknowledgements
Funding was provided by NIH/NIDCR R01-DE030716-01 and NIH/NIAMS R01-DE030716-01S1 to AS, NIH/NIAMS R01AR080131 to RG, and UConn REP Convergence Grant to RG and AS.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 10% Formalin Solution | Sigma-Aldrich | HT501128 | |
| 25 G 0.625 in Air-tite EZ Flo Hypodermic Needle | Ait Tite/ Fisher Scientific | 14-817-239; ref #NEZ25058 | |
| 26 G 1/2" Disposable Hypodermic Needles | Exel International/ Fisher Scientific | 14-840-83; ref #26402 | Any 26 G needles may be substituted, must be detachable from syringe |
| 3 mL Sterile Syringe | Fisherbrand/ Fisher Scientific | 14-955-457 | |
| AB Sterile Pad | McKesson/ Fisher Scientific | NC0593755 | Alternatively can use autoclavable absorbant pads |
| Acetic Acid, Glacial | Millipore Sigma | AX0073 | Needs to be diluted to 1% |
| Ammonium-Chloride-Potassium (ACK) Lysing Buffer | Gibco/ Thermo Fisher Scientific | A1049201 | Used for red blood cell lysis in preparation for flow cytometry |
| Anesthesia Induction Chamber 2 L | VetEquip | 941444 | |
| Betadine (Providone-Iodine) Solution | Penn Veterinary Supply Inc/ Fisher Scientific | NC0158124 | |
| Buprenorphine HCL Injection 0.3 mg/mL | Generic/ Covetrus | 59122 | Must obtain state and federal DEA license for purchasing controlled substance |
| Calcein-AM | Sigma Aldrich | 206700-1MG | |
| Collagenase P | Roche | 11249002001 | Used for digesting bone |
| Cotton Swabs and Applicators | Fisherbrand/ Fisher Scientific | 22-363-172 | |
| Cryofilm Type II C(10), 3.5 cm | Section Lab, Japan | CFS 105 | |
| DAPI Staining Solution | Abcam | ab228549 | |
| Digital Gram Scale | Toprime/ Amazon | B06X6LW4V9 | Can use any brand scale to weigh mice |
| Dissecting Forceps | Fisherbrand/ Fisher Scientific | 08-953E; 08-953F | |
| Dissecting Scissors | Fisherbrand/ Fisher Scientific | 08-940 | |
| Endure Low Profile Cryostat/Microtome Blades | Tanner Scientific | TNR315LP | |
| Ethiqa XR Buprenorphine Extended-Release 1.3 mg/mL | Ethiqa XR/ Cevetrus | 72117 | Must obtain state and federal DEA license for purchasing controlled substance |
| Fast Green, 0.2% | Electron Miscroscopy Sciences EMS | 50-319-57 | |
| Fetal Bovine Serum heat inactivated | Gibco/ Thermo Fisher Scientific | A5256801 | Used to make FACS Staining Media (FSM) |
| Fluoromount-G Mounting Solution | SouthernBiotech | 0100-01 | |
| GLUSEAL Topical Skin Adhesive | AD Surgical | GLU-550 | |
| Goat anti-Rabbit Alexa 647 | Thermo Fisher | A21244 | 1:300 dilution |
| Hanks' Balanced Salt Solution (HBSS) | Gibco/ Thermo Fisher Scientific | 14065056 | Used to make FACS Staining Media (FSM) |
| Heating Pad (large) | Boncare/ Amazon | B0CLHSWXSX | Can use any brand heating pad, remove the cloth cover |
| Hematoxylin | MilliporeSigma/ Fisher Scientific | M1051740500 | |
| Hyaluronidase | MilliporeSigma | HX0514 | Used for digesting bone |
| Isoflurane Animal Anesthesia System | VetEquip | 901810 | |
| KUBTEC Parameter 2D Cabinet X-ray System | OncoMed Solutions | N/A | Any X-ray Cabinet imaging system can be used |
| Microcentrifuge Tubes | Corning/ Thomas Scientific | 8600A52 | |
| Microscope Cover Glass, 18 mm x 18 mm, # 1 Thickness | Globe Scientific | 1414-10 | |
| N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid HEPES (1M) | Gibco/ Thermo Fisher Scientific | 156300-80 | Used to make FACS Staining Media (FSM) |
| Norland optical Adhesive NO61 | Edmund Optical | 37-322 | Used to adhere cryofilm sections to a slide |
| Osterix Rabbit Monoclonal Antibody | Abcam | ab209484 | 1:500 dilution |
| Peroxigard Ready to Use One Step Disinfectant | Virox Technologies | 29101 | Can be substituted with any another clinical/lab grade disinfectant |
| Propidium Iodide (PI) | Invitrogen/ Thermo Fisher Scientific | P1304MP | Nuclear fluorescent stain used to detect dead cells for vioability counts |
| Rechargeable Electric Trimmer | Wahl/ Amazon | B001FWXKUE | Any clippers can be used, a smaller sized trimmer will make it easier to shave smaller animals |
| Rodent Nose Cone | VetEquip | 921609 | |
| Safranin O, 85% pure, certified | Fisher Scientific | AC419211000 | |
| Seal'N Freeze Cryotray Square Molds | Electron Miscroscopy Sciences EMS | 62642-01 | Used for embedding, other embedding molds can be used. This specific model has a deeper mold, as well as a lid to seal the block |
| Silver Nitrate | Sigma Aldrich | S6506 | Silver nitrate for von Kossa stain |
| Sterile Phosphate Buffered Saline (PBS) | Gibco/ Thermo Fisher Scientific | 10010023 | Alternatively, freshly prepared PBS that has been filtered and autoclaved can be used |
| Sterile Standard Scalpels | Integra Miltex/ Fisher Scientific | 12-460-455 | |
| Sterile Standard Scalpels | Integra Miltex/ Fisher Scientific | 12-460-455 | |
| Sucrose Powder 1 KG | Thermo Fisher Scientific | 036508-A1 | |
| Superfrost Plus Miscroscope Slides | Fisherbrand/ Fisher Scientific | 1255015 | Adhesive slides |
| Sure-Tek Tissue Processing/ Embedding Casettes | Fisherbrand Histosette/ Fisher Scientific | 22-048-141 | |
| Tissue Plus Optimal Cutting Temperature O.C.T Compound | Fisher Scientific | 23-730-571 | |
| Tissue Sponges | Epredia/ Fisher Scientific | 84-53 | Tissue sponges protect tissues from overproocessing |
| Toluidine Blue | Fisher Scientific | AC348600050 |
References
- Bahney, C. S., et al. Cellular biology of fracture healing. J Orthop Res. 37 (1), 35-50 (2019).
- Ko, F. C., Sumner, D. R. How faithfully does intramembranous bone regeneration recapitulate embryonic skeletal development. Dev Dyn. 250 (3), 377-392 (202....
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