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.

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
<ol>
	<li>Prepare the surgical procedure room by disinfecting working surfaces with a clinical-grade disinfectant cleaner. 
	NOTE: The surgeon should wear appropriate personal protective equipment (PPE), including sterile disposable gloves, hair bonnet, gown, and mask.</li>
	<li>Prepare a heating pad covered with a sterile surgical pad. Gather and arrange all sterile instruments and reagents (Table of Materials) with convenient access.</li>
</ol>
2. Pre-operative anesthesia
<ol>
	<li>Weigh all mice before surgery. Calculate the volumes of analgesic to be used based on the available formulation of buprenorphine.</li>
	<li>Assemble an animal anesthesia unit within the sterile working space, which includes an induction chamber and a nose-cone assembly. Set oxygen output to 2 L min-1 and isoflurane to 1.5-3% (v/v) depending on mouse age and weight.</li>
	<li>Once the mouse is sedated, remove the animal from the induction chamber and place it on a sterile surgical pad in the supine position. Apply ophthalmic lubricant to both eyes. Place the nose cone over the head. Check that the mouse is adequately anesthetized by gently pinching the toe. Ensure there is no hindlimb reflex response before proceeding.</li>
	<li>Prior to surgery, briefly remove the mouse from nose cone to&#160;inject the total dose of extended-release buprenorphine via the subcutaneous route; the total dose is 3.25 mg/kg.</li>
</ol>
3. Surgical procedure description
NOTE: This surgical procedure utilizes C57BL/6 male and female mice aged 10 weeks to 24 weeks. The procedure can be performed on mice as young as 6 weeks up to 33 weeks old and on various inbred strains, as shown by others10,11. Younger mice may require shorter needle lengths, while older or larger mice may require a longer needle.
<ol>
	<li>Fill a 3 mL syringe equipped with a 26 G needle with sterile saline. Place this to the side.</li>
	<li>Remove the fur from the hindlimb on which procedure will be performed with electric or battery-operated clippers (#40 blade) and remove any loose fur from the surgical field. Using sterile cotton-tipped applicators, treat the surgical site with an iodine-based scrub (10% Povidone-iodine), followed by 70% isopropyl alcohol. Scrub the surgical site starting at the center of the knee and making a circular sweep outward. Allow the site to dry. Repeat the scrub steps 3 times.&#160;</li>
	<li>Place the mouse on its back and flex the knee of the operative leg. Hold the leg of the mouse in the non-dominant hand such that the knee is bent over the middle finger, the second finger rests on the femur, and the thumb rests on the tibia.</li>
	<li>Make a small, shallow horizontal incision just below the patella using a scalpel. 
	NOTE: This step improves visualization of the insertion site. With experience, this step can be skipped.</li>
	<li>Locate the joint space by placing the needle horizontally across the bent knee and feeling for the space between the femur and tibia. Using this anatomical site to guide, take a 25 G needle and manually drill (by twisting the needle and simultaneously applying moderate pressure onto the needle) into the distal femur from the knee toward the proximal side. Insert the needle through or around the patella.</li>
	<li>X-ray the mouse to ensure the needle has been appropriately placed in the medullary cavity, as close to the center as possible (Figure 1).</li>
	<li>Remove the 25 G needle and immediately insert a 26 G needle into the same hole within the marrow cavity. Ream the sides of the medullary cavity in a circular motion at least 5 times until it feels smooth. Keep the number of reaming motions consistent between each mouse. 
	NOTE: Steps 3.5 and 3.7 may cause needlestick injury to the surgeon if not handled carefully.</li>
	<li>Remove the 26 G needle and replace the 26 G needle connected to the saline syringe. Flush the cavity until the backflow runs clear. This typically requires 1.5-2 mL of saline. Remove the syringe and needle and safely dispose of them in a sharp container. 
	NOTE: There should not be resistance when pushing the fluid. If so, re-insert the needle to ensure it is within the cavity. It may be necessary to pull the needle out halfway.</li>
	<li>Close the skin wound using a topical adhesive suture.</li>
</ol>
4. Postoperative care
<ol>
	<li>Immediately following surgery, administer the remaining half dose of extended-release buprenorphine using dosing from step 2.4. 
	NOTE: Extended-release Buprenorphine provides pain relief for up to 72 h.</li>
	<li>Monitor the animals on the heating pad for signs of normal, unobstructed breathing until they awaken from surgery. Once ambulatory, return mice to their cage.</li>
	<li>Continue to monitor the mice once per day for 3 days after the surgery to ensure they are healing properly and have full mobility, watching for signs of pain.</li>
</ol>
5. Processing bones for &#181;CT scanning and analysis
<ol>
	<li>Euthanize the mice according to the humane euthanization method regulated and approved by the IACUC or other applicable institutional policy. Here, the primary euthanasia method of CO2 inhalation was used, followed by a secondary method of cervical dislocation.</li>
	<li>Harvest the femurs by peeling away the skin around the hindlimbs and removing the entire limb at the hip joint.</li>
	<li>Sandwich each limb between two sponges in an embedding cassette. Transfer the bones in their cassettes to a 10% formalin solution and fix them at 4 &#176;C for 5-7 days, considering the age and size of the mouse. 
	NOTE: Both over-fixation and under-fixation result in poor marrow resolution.</li>
	<li>After fixation, wash the cassettes in phosphate buffered saline (PBS) for 5 min 3 times.</li>
	<li>Remove the limbs from the cassettes and trim away muscle using a scalpel as needed. 
	NOTE: Depending on downstream applications, rough handling can disrupt the periosteal layer of bone.</li>
	<li>Separate the femur at the knee by carefully cutting through the ligaments using small surgical scissors or a scalpel. 
	NOTE: The tibias can be used for other applications or discarded.</li>
	<li>Transfer the femurs to 1.7 mL or 15 mL conical tubes into a 70% ethanol solution. Bones can remain in 70% ethanol at 4 &#176;C until ready for &#181;CT scanning.</li>
	<li>Scan and assess the femurs for trabecular parameters, producing qualitative and quantitative data.</li>
</ol>
6. Processing bones for frozen sectioning and histology
NOTE: Bones can be processed for frozen sectioning and histology after undergoing &#181;CT scanning. The scanning does not affect fluorescent labeling or subsequent antibody staining.
<ol>
	<li>Transfer the bones from 70% ethanol to PBS and wash 3 times, 5 min per wash.</li>
	<li>Incubate in a series of sucrose solutions from 10%, 20%, to 30% in PBS at 4 &#176;C overnight at each concentration. Store the bones in 30% sucrose for up to 5 days at 4 &#176;C or in this solution at -20 &#176;C for 6-12 months until ready for embedding. 
	NOTE: Using a sucrose gradient results in better tissue penetration. After step 6.1, bones may be placed directly into a 30% sucrose solution overnight.</li>
	<li>Embed bones in optimal cutting temperature (OCT) compound using cryoembedding molds. Store at -80 &#176;C until ready to section.</li>
	<li>Section embedded bones using the cryofilm tape method with cryostat temperature set between -24 &#176;C and -26 &#176;C.</li>
	<li>Adhere the sections on the cryofilm to an adhesive slide using optical adhesive and cure it until hardened using an ultraviolet (UV) crosslinker.</li>
</ol>
7. Processing for flow cytometry
NOTE: This protocol is compatible with a variety of antibodies. For characterizing skeletal stem and progenitor cells, pooling 3-4 bones together is recommended as these are rare cell populations in the bone marrow.
<ol>
	<li>Prepare fluorescence-activated cell sorting (FACS) staining media (FSM). To make 1000 mL solution of FSM, combine: 100 mL of Hanks&#39; balanced salt solution (HBSS), 10 mL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 20 mL of Fetal bovine serum (FBS), 870 mL of dd H2O. Pass the FSM solution through a 0.2 &#181;m filter.</li>
	<li>Before harvesting tissue, fill Petri dishes with 4-5 mL of sterile PBS without calcium and magnesium.</li>
	<li>Prepare the digestion media
	<ol>
		<li>For every 4-6 femurs, prepare a digestion mixture in 10 mL of PBS containing 0.005 g of collagenase P and 0.02 g of hyaluronidase. Mix by gentle vortexing and pass the mixture through a 0.22 &#181;m filter.</li>
	</ol>
	</li>
	<li>Harvest bones on post-surgery Day 3 as described in step 5.2. Remove all muscle and connective tissue with a scalpel blade.</li>
	<li>Fill a Petri dish with 5 mL of digestion media. Place 4-6 bones in the digestion media. Chop each bone finely, making the first cut longitudinally and laterally.</li>
	<li>Transfer tissue to a 14 mL round-bottom tube and seal with parafilm. Cover tubes in aluminum foil.</li>
	<li>Shake at 37 &#176;C for 20 min by keeping the tube horizontally on the shaker.</li>
	<li>Remove the tube from the shaker. Cells will be clumped at the bottom. Gently collect the digested material without disturbing the cells at the bottom, and pass the digest through a 70 &#181;m filter. Collect the filtrate in a new 50 mL tube containing 10 mL of cold FSM and keep it on ice.</li>
	<li>To the cells, add the remaining 5 mL of the digestion mix. Seal again with parafilm and cover with aluminum foil. Shake the tube as described above. Repeat steps 7.7-7.8.</li>
	<li>After the 20 min incubation at 37 &#176;C, transfer the digest to the same tube containing the digest from step 7.8, passing through a 70 &#181;m filter with another 10 mL of FSM.</li>
	<li>Centrifuge the suspension at 274 x g at 4 &#176;C. Aspirate the supernatant and collect the cell pellet. Dislodge the pellet by gently tapping the tube. 
	NOTE: The centrifugal force (g) is based on a rotor radius of 170 mm. Please check the rotor radius and adjust the speed accordingly.</li>
	<li>To perform the red blood cell lysis, add 1 mL of ammonium-chloride-potassium (ACK) lysing buffer. Keep all reagents and cells on ice.</li>
	<li>Incubate the cells in ACK for about 5 min at RT. Do not exceed 5 min to avoid overly lysing the cells.</li>
	<li>Add 9 mL of FSM. Centrifuge at 274 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant. Break up the cell pellet by gently raking the tube on a tube rack.</li>
	<li>Add 10 mL of FSM (volume for 4 bones, can be adjusted based on how many bones are combined) and resuspend.</li>
	<li>Take an aliquot of each sample and add propidium iodide (PI) as per the manufacturer&#39;s instructions. Count live and total cells with a cell counter.</li>
	<li>Proceed with antibody staining of samples using desired markers for flow cytometry analysis12,13,14.</li>
</ol>

Improved Bone Marrow Injury Model for Intramembranous Bone Repair Studies

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The authors have no disclosures to report.

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...

Long&#160;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.&#160;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. Interestingly, in studies of patients with long bone fractures, 66%-82% had displaced fractures, which required fixation devices to stabilize the bone4,5. These rigidly stabilized bones primarily heal through intramembranous ossification as the broken bone ends come in direct contact with each other6,7. Yet, the mechanistic regulation of intramembranous bone regeneration remains relatively understudied compared to bone formation via the endochondral pathways. Food and Drug Administration (FDA)-approved therapies to augment bone healing are hindered by limited clinical efficacy and high costs and are associated with significant adverse effects8.
Mechanical bone marrow ablation provides a valuable model for studying intramembranous bone formation. This simple injury model allows for studying bone regeneration in the bone marrow without disrupting the cortical bone. Following BM ablation, the healing process involves distinct yet overlapping phases. The initial phase occurs in the first 1 to 5 days and involves clot formation and inflammation, where inflammatory cells and cytokines initiate healing. From days 3 to 14, the second phase is characterized by regeneration, including neovascularization, mesenchymal stem cell (MSC) migration and proliferation, osteoblastic differentiation, and woven bone formation. The final remodeling phase begins approximately 10 days after surgery, with bone tissue undergoing maturation and restructuring until the marrow is fully restored by day 569. This rapid healing timeline makes the BM ablation model ideal for studying early bone repair responses, particularly during the critical periods of inflammation, progenitor cell recruitment, and osteoblast differentiation. Using techniques like histology, flow cytometry, and quantitative &#181;CT analysis, cellular responses and bone formation in early repair stages can be evaluated. Using the aforementioned techniques, the BM ablation model can provide insight into mechanisms of intramembranous bone regeneration and aid in identifying key therapeutic targets for enhancing bone healing.

<table><tbody><tr><td>10% Formalin Solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>25 G 0.625 in Air-tite EZ Flo Hypodermic Needle</td><td>Ait Tite/ Fisher Scientific</td><td>14-817-239; ref #NEZ25058</td><td></td></tr><tr><td>26 G 1/2&quot; Disposable Hypodermic Needles</td><td>Exel International/ Fisher Scientific</td><td>14-840-83; ref #26402</td><td>Any 26 G needles may be substituted, must be detachable from syringe&amp;nbsp;</td></tr><tr><td>3 mL Sterile Syringe</td><td>Fisherbrand/ Fisher Scientific&amp;nbsp;</td><td>14-955-457</td><td></td></tr><tr><td>AB Sterile Pad</td><td>McKesson/ Fisher Scientific</td><td>NC0593755</td><td>Alternatively can use autoclavable absorbant pads&amp;nbsp;</td></tr><tr><td>Acetic Acid, Glacial</td><td>Millipore Sigma</td><td>AX0073</td><td>Needs to be diluted to 1%</td></tr><tr><td>Ammonium-Chloride-Potassium (ACK) Lysing Buffer</td><td>Gibco/ Thermo Fisher Scientific&amp;nbsp;</td><td>A1049201</td><td>Used for red blood cell lysis in preparation for flow cytometry&amp;nbsp;</td></tr><tr><td>Anesthesia Induction Chamber 2 L</td><td>VetEquip</td><td>941444</td><td></td></tr><tr><td>Betadine (Providone-Iodine) Solution&amp;nbsp;</td><td>Penn Veterinary Supply Inc/ Fisher Scientific</td><td>NC0158124</td><td></td></tr><tr><td>Buprenorphine HCL Injection 0.3 mg/mL&amp;nbsp;</td><td>Generic/ Covetrus</td><td>59122</td><td>Must obtain state and federal DEA license for purchasing controlled substance</td></tr><tr><td>Calcein-AM</td><td>Sigma Aldrich</td><td>206700-1MG</td><td></td></tr><tr><td>Collagenase P&amp;nbsp;</td><td>Roche</td><td>11249002001</td><td>Used for digesting bone</td></tr><tr><td>Cotton Swabs and Applicators</td><td>Fisherbrand/ Fisher Scientific&amp;nbsp;</td><td>22-363-172</td><td></td></tr><tr><td>Cryofilm Type II C(10), 3.5 cm</td><td>Section Lab, Japan</td><td>CFS 105</td><td></td></tr><tr><td>DAPI Staining Solution&amp;nbsp;</td><td>Abcam</td><td>ab228549</td><td></td></tr><tr><td>Digital Gram Scale</td><td>Toprime/ Amazon</td><td>B06X6LW4V9</td><td>Can use any brand scale to weigh mice</td></tr><tr><td>Dissecting Forceps&amp;nbsp;</td><td>Fisherbrand/ Fisher Scientific&amp;nbsp;</td><td>08-953E; 08-953F</td><td></td></tr><tr><td>Dissecting Scissors&amp;nbsp;</td><td>Fisherbrand/ Fisher Scientific&amp;nbsp;</td><td>08-940</td><td></td></tr><tr><td>Endure Low Profile Cryostat/Microtome Blades</td><td>Tanner Scientific</td><td>TNR315LP</td><td></td></tr><tr><td>Ethiqa XR Buprenorphine Extended-Release 1.3 mg/mL</td><td>Ethiqa XR/ Cevetrus</td><td>72117</td><td>Must obtain state and federal DEA license for purchasing controlled substance</td></tr><tr><td>Fast Green, 0.2%</td><td>Electron Miscroscopy Sciences EMS</td><td>50-319-57</td><td></td></tr><tr><td>Fetal Bovine Serum heat inactivated</td><td>Gibco/ Thermo Fisher Scientific&amp;nbsp;</td><td>A5256801</td><td>Used to make FACS Staining Media (FSM)</td></tr><tr><td>Fluoromount-G Mounting Solution&amp;nbsp;</td><td>SouthernBiotech&amp;nbsp;</td><td>0100-01</td><td></td></tr><tr><td>GLUSEAL Topical Skin Adhesive&amp;nbsp;</td><td>AD Surgical&amp;nbsp;</td><td>GLU-550</td><td></td></tr><tr><td>Goat anti-Rabbit Alexa 647</td><td>Thermo Fisher</td><td>A21244</td><td>1:300 dilution</td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS)</td><td>Gibco/ Thermo Fisher Scientific</td><td>14065056</td><td>Used to make FACS Staining Media (FSM)</td></tr><tr><td>Heating Pad (large)</td><td>Boncare/ Amazon</td><td>B0CLHSWXSX</td><td>Can use any brand heating pad, remove the cloth cover&amp;nbsp;</td></tr><tr><td>Hematoxylin</td><td>MilliporeSigma/ Fisher Scientific</td><td>M1051740500</td><td></td></tr><tr><td>Hyaluronidase</td><td>MilliporeSigma</td><td>HX0514</td><td>Used for digesting bone</td></tr><tr><td>Isoflurane Animal Anesthesia System</td><td>VetEquip</td><td>901810</td><td></td></tr><tr><td>KUBTEC Parameter 2D Cabinet X-ray System</td><td>OncoMed Solutions</td><td>N/A</td><td>Any X-ray Cabinet imaging system can be used</td></tr><tr><td>Microcentrifuge Tubes</td><td>Corning/ Thomas Scientific&amp;nbsp;</td><td>8600A52</td><td></td></tr><tr><td>Microscope Cover Glass, 18 mm x 18 mm, # 1 Thickness</td><td>Globe Scientific</td><td>1414-10</td><td></td></tr><tr><td>N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid HEPES (1M)&amp;nbsp;</td><td>Gibco/ Thermo Fisher Scientific</td><td>156300-80</td><td>Used to make FACS Staining Media (FSM)</td></tr><tr><td>Norland optical Adhesive NO61</td><td>Edmund Optical</td><td>37-322</td><td>Used to adhere cryofilm sections to a slide&amp;nbsp;</td></tr><tr><td>Osterix Rabbit Monoclonal Antibody&amp;nbsp;</td><td>Abcam</td><td>ab209484</td><td>1:500 dilution</td></tr><tr><td>Peroxigard Ready to Use One Step Disinfectant</td><td>Virox Technologies</td><td>29101</td><td>Can be substituted with any another clinical/lab grade disinfectant</td></tr><tr><td>Propidium Iodide (PI)&amp;nbsp;</td><td>Invitrogen/ Thermo Fisher Scientific&amp;nbsp;</td><td>P1304MP</td><td>Nuclear fluorescent stain used to detect dead cells for vioability counts</td></tr><tr><td>Rechargeable Electric Trimmer</td><td>Wahl/ Amazon</td><td>B001FWXKUE</td><td>Any clippers can be used, a smaller sized trimmer will make it easier to shave smaller animals</td></tr><tr><td>Rodent Nose Cone</td><td>VetEquip</td><td>921609</td><td></td></tr><tr><td>Safranin O, 85% pure, certified</td><td>Fisher Scientific</td><td>AC419211000</td><td></td></tr><tr><td>Seal&#039;N Freeze Cryotray Square Molds</td><td>Electron Miscroscopy Sciences EMS</td><td>62642-01</td><td>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&amp;nbsp;</td></tr><tr><td>Silver Nitrate</td><td>Sigma Aldrich</td><td>S6506</td><td>Silver nitrate for von Kossa stain</td></tr><tr><td>Sterile Phosphate Buffered Saline (PBS)</td><td>Gibco/ Thermo Fisher Scientific</td><td>10010023</td><td>Alternatively, freshly prepared PBS that has been filtered and autoclaved can be used</td></tr><tr><td>Sterile Standard Scalpels</td><td>Integra Miltex/ Fisher Scientific</td><td>12-460-455</td><td></td></tr><tr><td>Sterile Standard Scalpels</td><td>Integra Miltex/ Fisher Scientific</td><td>12-460-455</td><td></td></tr><tr><td>Sucrose Powder 1 KG</td><td>Thermo Fisher Scientific</td><td>036508-A1</td><td></td></tr><tr><td>Superfrost Plus Miscroscope Slides</td><td>Fisherbrand/ Fisher Scientific&amp;nbsp;</td><td>1255015</td><td>Adhesive slides</td></tr><tr><td>Sure-Tek Tissue Processing/ Embedding Casettes</td><td>Fisherbrand Histosette/ Fisher Scientific</td><td>22-048-141</td><td></td></tr><tr><td>Tissue Plus Optimal Cutting Temperature O.C.T Compound</td><td>Fisher Scientific</td><td>23-730-571</td><td></td></tr><tr><td>Tissue Sponges</td><td>Epredia/ Fisher Scientific</td><td>84-53</td><td>Tissue sponges protect tissues from overproocessing&amp;nbsp;</td></tr><tr><td>Toluidine Blue</td><td>Fisher Scientific</td><td>AC348600050</td><td></td></tr></tbody></table>

improved methodology for studying postnatal osteogenesis via intramembranous ossification in a murine bone marrow injury model

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 (&#181;CT) 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.

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...

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 ...

improved-methodology-for-studying-postnatal-osteogenesis-via

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

259 Views.  UConn School of Medicine. 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. 

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

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7, after application of bioactive molecules 8,9, after altering physiological parameters of healing 10, after modifying the amount or time of stabilization 11, after distraction osteogenesis 12, after creation of a non-union 13, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.

This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators 1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role ...

Medicine

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Developmental Biology

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.

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.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...

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

Summary

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Chapters in this video

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

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

A Simple Critical-sized Femoral Defect Model in Mice

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Establishing a Diaphyseal Femur Fracture Model in Mice