We thank members of the Muneoka Lab and the Texas Institute for Genomic Medicine (TIGM). This work was supported by Texas A&#38;M University.

All animal use and techniques were in compliance with the standard operating procedures of the Institutional Animal Care and Use Committee of Texas A&#38;M University.
1. Adult Mouse Hind Limb Distal P3 Amputation
<ol>
	<li>Anesthetize an 8 to 12 week-old CD-1 mouse (Table of Materials) using isoflurane gas in oxygen; initially anesthetize at 3% in a chamber, followed by 2% isoflurane supplied by a nosecone over the duration of the surgery. Apply ophthalmic ointment on eyes ...

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P., 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=Axonal+regrowth+is+impaired+during+digit+tip+regeneration+in+mice.">Axonal regrowth is impaired during digit tip regeneration in mice.</a> Developmental Biology. 445, 237-244 (2018).</li><li>Dawson, L. 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=Blastema+formation+and+periosteal+ossification+in+the+regenerating+adult+mouse+digit.">Blastema formation and periosteal ossification in the regenerating adult mouse digit.</a> Wound Repair and Regeneration: Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. 26, 263-273 (2018).</li><li>Sammarco, M. C., 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=Endogenous+bone+regeneration+is+dependent+upon+a+dynamic+oxygen+event.">Endogenous bone regeneration is dependent upon a dynamic oxygen event.</a> Journal of Bone and Mineral Research: The Official Journal of the American Society for Bone and Mineral Research. 29, 2336-2345 (2014).</li><li>Einhorn, T. 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A., Muneoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digit+Tip+Regeneration:+Merging+Regeneration+Biology+with+Regenerative+Medicine.">Digit Tip Regeneration: Merging Regeneration Biology with Regenerative Medicine.</a> Stem Cells Translational Medicine. 7, 262-270 (2018).</li><li>Lee, J., 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=SDF-1alpha/CXCR4+signaling+mediates+digit+tip+regeneration+promoted+by+BMP-2.">SDF-1alpha/CXCR4 signaling mediates digit tip regeneration promoted by BMP-2.</a> Developmental Biology. 382, 98-109 (2013).</li><li>Doube, M., 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=BoneJ:+Free+and+extensible+bone+image+analysis+in+ImageJ.">BoneJ: Free and extensible bone image analysis in ImageJ.</a> Bone. 47, 1076-1079 (2010).</li></ol>

This protocol describes a standardized procedure of adult mouse distal P3 amputation, fluorescent immunohistochemical staining to visualize and investigate blastema formation and intramembranous ossification, and sequential in-vivo &#181;CT scanning to identify bone morphological, volume, and length changes post amputation. P3 amputation is a unique, procedurally simple, and reproducible model to analyze a pro-regenerative wound environment that triggers blastema formation. Furthermore, the P3 digit model offers numerous...

Mammals, including humans and mice, have the capacity to regenerate the tips of their digits after distal amputation of the terminal phalanx (P3)1,2,3. In mice, the regeneration response is amputation-level-dependent; increasingly proximal digit amputations display a progressively attenuated regenerative response until complete regenerative failure at amputations transecting and proximal to the P3 nail matrix4,5,6,7,8. P3 regeneration is mediated by the formation of a blastema, defined as a population of proliferating cells that undergo morphogenesis to regenerate the amputated structures9. The formation of a blastema to regenerate the structures lost by amputation, a process termed epimorphic regeneration, distinguishes the multi-tissue-level P3 regeneration response from traditional tissue repair after injury6,10. P3 regeneration is a reproducible and procedurally simple model to investigate complex regenerative processes including wound healing11,12, bone histolysis11,12, revascularization13, peripheral nerve regeneration14, and blastemal conversion to bone via intramembranous ossification15.
Previous studies using immunohistochemistry have demonstrated that the blastema is heterogeneous, avascular, hypoxic, and highly proliferative11,13,15,16. Following distal P3 amputation, the early blastema is initially associated with the P3 periosteum and endosteum and is characterized by robust proliferation and nascent osteogenesis adjacent to the bone surface15. Subsequent to bone degradation and wound closure, the heterogeneous blastema is formed by the merging of periosteal and endosteal-associated cells, followed by the differentiation of blastemal components including bone via intramembranous ossification15.
Bone repair in response to injury typically occurs by endochondral ossification, i.e. via an initial cartilaginous callus that forms a template for subsequent bone formation17,18. Long bone intramembranous ossification, i.e., bone formation without a cartilaginous intermediate, is commonly induced using complex distraction devices or surgical fixation19,20. The digit regeneration response is a pre-clinical model that offers advantages over conventional intramembranous ossification models: 1) it does not require external or internal fixation post injury to stimulate intramembranous ossification, 2) it is performed using 4 digits from each animal, thus maximizing samples while minimizing animal use, and 3) sequential in vivo microcomputed tomography (&#956;CT) analysis can be performed with ease and speed.
In the present study, we show the standardized P3 amputation plane to achieve a reproducible and robust regeneration response. Additionally, we demonstrate an optimized fluorescence immunohistochemistry protocol using paraffin sections to visualize blastema formation, revascularization in the context of regeneration, and blastemal conversion to bone via intramembranous ossification. We also demonstrate the use of sequential in-vivo &#956;CT to identify changes in bone morphology, volume, and length in the same digit over the course of regeneration. The goal of this protocol is to investigate mammalian blastema formation after amputation and to demonstrate 2 techniques, fluorescence immunohistochemistry and sequential in vivo &#956;CT, for the study of intramembranous bone regeneration.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protein Block Serum Free</td>
			<td>DAKO</td>
			<td>X0909</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Mouse anti-PCNA antibody</td>
			<td>Abcam</td>
			<td>ab29</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>Rat anti-CXCR4 antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB21651</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Rabbit anti-human vWF XIII antibody</td>
			<td>DAKO</td>
			<td>A0082</td>
			<td>1:800 dilution</td>
		</tr>
		<tr>
			<td>Rabbit anti-osterix, SP7 antibody</td>
			<td>Abcam</td>
			<td>ab22552</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Rabbit anti-Runx2 antibody</td>
			<td>Sigma-Aldrich Co.</td>
			<td>HPA022040</td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-conjugated goat anti-mouse IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A21235</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568-conjugated goat anti-rat IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A11077</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Prolong Gold antifade reagent</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Surgipath Decalicifier 1</td>
			<td>Leica Biosystems</td>
			<td>3800400</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Z-Fix, Aqueous buffered zinc formalin fixative</td>
			<td>Anatech LTD</td>
			<td>174</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>CD-1 Female Mouse</td>
			<td>Envigo</td>
			<td>ICR(CD-1)</td>
			<td>8-12-weeks-old</td>
		</tr>
		<tr>
			<td>vivaCT 40</td>
			<td>SCANCO Medical</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

adult mouse digit amputation and regeneration: a simple model to investigate mammalian blastema formation and intramembranous ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Adult mouse regenerating P3 digits at 6/7 DPA (Figure 2A-D), 9 DPA (Figure 2E-H), and 10 DPA (Figure 2I-L) were immunostained with antibodies to Runx2, OSX, and PCNA to visualize intramembranous bone regeneration, and immunostained with antibodies to CXCR4 and vWF to visualize blastema formation. Representative &#956;CT renderings of digits scanned prior to amputation and at various...

Watch this Scientific Journal Video about Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification at JoVE.com

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

P3 amputation is a preclinical model for investigating mammalian epimorphic regeneration and a powerful tool for identifying critical cell populations and mechanisms that control endogenous regeneration. The P3 model allows for the identification of early and late blastemal cell populations, the study of revascularization during regeneration, and the investigation of intramembranous ossification without complex bone stabilization device requirements. Demonstrating the procedure with me will be Osama Qureshi, Alyssa Falck, and Katherine Zimmel, technician, and Regina Brunaeur, a Research Assistant Professor all from our laboratory.After confirming a lack of response to toe pinch in an eight to 12-week-old CD-1 mouse, Apply ointment to the animal's eyes and place the mouse under a 10X dissection microscope. Use surgical povidone iodine and 70%ethanol to sterilize the digits of each hind limb and use microscissors to trim hair away from the surgical site. Apply one microliter of topical bupivacaine locally to anesthetize the surgical site, and gently splay one hind paw to expose the medial digit surface.Then holding the scalpel at an angle parallel to the fat pad, use a sterile number 10 scalpel to amputate the distal tip of the terminal phalanx of digits two and four. Apply one additional microliter of bupivacaine locally to each amputated digit and return the mouse to a clean cage to allow the wounds to heal without wound dressing and with monitoring until full recumbency. To assure a successful P3 amputation plane, hold the scalpel parallel to the fat pad during the amputation and perform micro-CT scanning to confirm the correct amputation length.At the appropriate experimental endpoint, use a scalpel to sever the digit midway through the middle phalanx bone at approximately the second ventral fat pad indent. Transfer the tissue to a 20-milliliter scintillation vial containing 10 milliliters of fresh buffered zinc formalin fixative for 24 to 48 hours. And use a microtome to acquire four to five micrometer thick sections of each digit, placing the slices into a 38 to 41 degrees Celsius water bath supplemented with a histological adhesive solution as they are obtained.Then capture the slices on adhesive slides and place the slides on a 37-degree Celsius slide warmer to dry. When the slides have been deparaffinized immerse the samples in a staining jar of an appropriate antigen retrieval solution, and heat the slides to 95 degrees Celsius for 25 minutes making sure that boiling does not occur. At the end of the incubation, allow the jar to come to room temperature, and lay the slides flat in a one-inch deep covered plastic slide box with moistened tissue paper at the base.Add 100 microliters of prewarmed proteinase K to each sample. Cover each slide with a small strip of parafilm to ensure the sections do not become dry. After 12 minutes at 37 degrees Celsius, carefully remove the paraffin film, and gently drain the slides on tissue paper to remove excess blocking solution.Next, add 100 to 200 microliters of the primary antibody solution of interest to each slide. Return the sites to the humidifying chamber covered with paraffin film for an overnight incubation at four degrees Celsius. The next morning after washing the slides in TRIS-buffered saline plus TWEEN or TBST label the slides with an appropriate secondary antibody solution for a 45-minute incubation at room temperature protected from light.At the end of the incubation, wash the slides in TBST and allow them to dry. Then use 100 microliters of anti-fade mounting medium to carefully place coverslips onto the dried slides and store the slides flat at room temperature overnight protected from light to allow the mounting medium to dry. For sequential in vivo micro-computed tomography or micro-CT outfit the micro-CT imager with tubing to enable oxygen flow into and out of the micro-CT animal chamber, making sure that the outflowing oxygen is equipped with an activated charcoal filter to trap the isoflurane.Set the Voxelsize to 10.5 micrometers at 55 peak kilovoltage and 145 microamps with 1, 000 projections per 180 degrees and continuous rotation with an Integration Time of 200 milliseconds, resulting in a maximum of 213 slices per scan. When the scanner is ready, gently place the anesthetized mouse in the micro-CT animal chamber with the hind limb digits arranged in flat, close association ventral side up with the left paw on the left side and the right on the right side of the scanning platform. Then apply surgical tape to gently secure the digits in place.Apply ophthalmic ointment to the animal's eyes before initiating the scan. After scanning, return animal to a clean cage with monitoring until full recumbency, and allow up to several hours for micro-CT image reconstruction. For analysis of the reconstructed 3D bone image, using the software provided with the micro-CT, convert the image files to a series of DICOM files.When all of the files have been converted, download the DICOM series in a single folder using a batch file downloader in a web browser. Load the BoneJ plug-in into the ImageJ plug-in folder. Drag-and-drop the DICOM file series folder into the ImageJ bar to open the files.A 16-bit image stack displaying all of the gray values will be opened. To display bone only, click Image Adjust and Threshold. Slide the upper threshold bar to the far right and adjust the lower threshold bar until only the bone is highlighted red.The lower threshold value will be approximately 10, 000 to 13, 000 at a maximal signal intensity of 32, 767. Click Apply and in the new dialog box, click Black background. Next, click OK.An eight-bit image displaying black and white values will be generated.With the white corresponding to bone. Draw a rectangle around the P3 bone to select it and duplicate the stack. To generate a 3D image, click Plugins and 3D Viewer and use the freehand selection tool to circle the bone to be deleted.Then right-click and select Fill selection to crop any unnecessary bone from the image. To quantify the bone volume from the 3D rendering, open the BoneJ plug-in and select Volume Fraction. A results window will appear and the bone volume displayed in millimeters cubed.To quantify the bone length from the 3D rendering, use the ImageJ multipoint tool. To capture an image of the 3D rendering, click View and take a snapshot. Then save the image as a TIF or JPEG file.In these images sections of an adult mouse regenerating P3 digit were immunostained with antibodies to visualize intramembranous bone regeneration and blastema formation at day six to seven, nine and 10 post-amputation. Here, representative micro-CT renderings of digits scanned prior to amputation and at various time points over the course of regeneration with landmarks used to identify length measurements are shown. The mouse digit model is a powerful system for analyzing a pro-regenerative wound environment that triggers blastema formation when amputated at a distal level.Conversely, proximal amputation of the digits serves as a model system for investigating regenerative failure as well a site for testing strategies for enhancing regeneration.

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8.5K Views.  Texas A&M University. P3 amputation is a preclinical model for investigating mammalian epimorphic regeneration and a powerful tool for identifying critical cell populations and mechanisms that control endogenous regeneration. The P3 model allows for the identification of early and late blastemal cell populations, the study of revascularization during regeneration, and the investigation of intramembranous ossification without complex bone stabilization device requirements. Demonstrating the procedure with me will b...