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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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+regeneration+after+local+injury:+a+review.">Bone marrow regeneration after local injury: a review.</a> Exp Hematol. 3 (2), 135-148 (1975).</li><li>Chitnis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+bone+fractures:+treatment+patterns+and+factors+contributing+to+use+of+intramedullary+nailing.">Long bone fractures: treatment patterns and factors contributing to use of intramedullary nailing.</a> Expert Rev Med Devices. 17 (7), 731-737 (2020).</li><li>Singaram, S., Naidoo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physical+impact+of+long+bone+fractures+on+adults+in+KwaZulu-Natal.">The physical impact of long bone fractures on adults in KwaZulu-Natal.</a> S Afr J Physiother. 76 (1), 1393 (2020).</li><li>Kim, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthopedic+implants+and+devices+for+bone+fractures+and+defects:+past,+present+and+perspective.">Orthopedic implants and devices for bone fractures and defects: past, present and perspective.</a> Eng Regen. 1, 6-18 (2020).</li><li>Marsell, R., Einhorn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+fracture+healing.">The biology of fracture healing.</a> Injury. 42 (6), 551-555 (2011).</li><li>Katagiri, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=morphogenetic+protein-induced+heterotopic+bone+formation:+what+have+we+learned+from+the+history+of+a+half+century.">morphogenetic protein-induced heterotopic bone formation: what have we learned from the history of a half century.</a> Jpn Dent Sci Rev. 51 (2), 42-50 (2015).</li><li>Wise, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+gene+expression+profiling+during+rat+femoral+marrow+ablation-induced+intramembranous+bone+regeneration.">Temporal gene expression profiling during rat femoral marrow ablation-induced intramembranous bone regeneration.</a> PLoS One. 5 (10), e12987 (2010).</li><li>Chen, X. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biglycan-deficient+mice+have+delayed+osteogenesis+after+marrow+ablation.">Biglycan-deficient mice have delayed osteogenesis after marrow ablation.</a> Calcified Tissue Int. 72 (5), 577-582 (2003).</li><li>Moran, M. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% Formalin Solution&#160;</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&#34; 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&#160;</td>
		</tr>
		<tr>
			<td>3 mL Sterile Syringe</td>
			<td>Fisherbrand/ Fisher Scientific&#160;</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&#160;</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&#160;</td>
			<td>A1049201</td>
			<td>Used for red blood cell lysis in preparation for flow cytometry&#160;</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&#160;</td>
			<td>Penn Veterinary Supply Inc/ Fisher Scientific</td>
			<td>NC0158124</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine HCL Injection 0.3 mg/mL&#160;</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&#160;</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&#160;</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&#160;</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&#160;</td>
			<td>Fisherbrand/ Fisher Scientific&#160;</td>
			<td>08-953E; 08-953F</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scissors&#160;</td>
			<td>Fisherbrand/ Fisher Scientific&#160;</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&#160;</td>
			<td>A5256801</td>
			<td>Used to make FACS Staining Media (FSM)</td>
		</tr>
		<tr>
			<td>Fluoromount-G Mounting Solution&#160;</td>
			<td>SouthernBiotech&#160;</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>GLUSEAL Topical Skin Adhesive&#160;</td>
			<td>AD Surgical&#160;</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&#39; 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&#160;</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&#160;</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)&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Osterix Rabbit Monoclonal Antibody&#160;</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)&#160;</td>
			<td>Invitrogen/ Thermo Fisher Scientific&#160;</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&#39;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&#160;</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&#160;</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&#160;</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

Research

JoVE Journal

Biology

224 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

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the proplatelet formation using in vitro-differentiated megakaryocytes; however, there is an increasing evidence that conventional culture systems do not faithfully recapitulate the differentiation/maturation process that takes places inside the bone marrow. In this manuscript, we present an explant method initially described in 1956 by Thi&#233;ry and Bessis to visualize megakaryocytes which have matured in their native environment, thus circumventing potential artifacts and misinterpretations. Fresh bone marrows are collected by flushing the femurs of mice, sliced into 0.5 mm cross sections, and placed in an incubation chamber at 37 &#176;C containing a physiological buffer. Megakaryocytes become gradually visible at the explant periphery and are observed up to 6 hours under an inverted microscope coupled to a video camera. Over time, megakaryocytes change their shape, with some cells having a spherical form and others developing thick extensions or extending many thin proplatelets with extensive branching. Both qualitative and quantitative investigations are carried out. This method has the advantage of being simple, reproducible, and fast as numerous megakaryocytes are present, and classically half of them form proplatelets in 6 hours compared to 4 days for cultured mouse megakaryocytes. In addition to the study of mutant mice, an interesting application of this method is the straightforward evaluation of the pharmacological agents on the proplatelet extension process, without interfering with the differentiation process that may occur in cultures.

Here, we detail the bone marrow explant method, from sample preparation to microscopic slide analysis, to evaluate the ability of megakaryocytes which have differentiated in their physiological environment to form proplatelets.

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the ...

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As ...