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

Summary

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.

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 implants^2,3. I....

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

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....

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.......

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
Aseptic Control Envirocide Disinfectant Cleaner	Aseptic Control Products/ Fisher Scientific	500432411	Can use any clincial grade disinfectant solution
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
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