Improved Methodology for Studying Postnatal Osteogenesis via Intramembranous Ossification in a Murine Bone Marrow Injury Model

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.

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.

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 implants^2,3. I....

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

1. Prepare the surgical procedure room by disinfecting working surfaces with 10% Lysol or other clinical-grade disinfectant cleaner.
   NOTE: The surgeon should wear appropriate per....

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.......

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.......

The authors have no disclosures to report.

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.