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 to prevent dryness while under anesthesia. 
	NOTE: The adult P3 amputation studies standardized in our lab are performed on 8 to 12 week-old mice, and the regeneration response is conserved in all tested strains.</li>
	<li>Under a 10x dissection microscope, sterilize the digits of the hind limb with surgical Povidone-iodine and 70% ethanol. Trim hair away from the surgical site using micro-scissors. Apply 1 &#181;L of topical Bupivacaine locally to anesthetize the surgical site.</li>
	<li>Amputate the distal tip of the terminal phalanx (P3) on digits 2 and 4 of each hind limb using a sterile #10 scalpel (Figure 1A). Amputation must be performed under aseptic conditions, including sterilization of surgical instruments. Under the dissection microscope, gently splay the hind paw to expose the medial digit surface. Hold the scalpel at an angle parallel to the fatpad to perform the amputation shown in Figure 1B.&#160;Apply 1 &#181;L of topical Bupivacaine to the amputation site. 
	NOTE: Amputation transects the nail organ, dermis, the P3 bone, vasculature, and nerves, but does not transect the marrow cavity or the digit fat pad. If bleeding is observed, apply direct pressure using a sterile cotton tip applicator.</li>
	<li>The standard P3 distal amputation plane removes approximately 15%&#8211;20% of the P3 bone volume21. MicroCT scanning (see below) can be performed to confirm the correct amputation length.</li>
	<li>Return the mouse to a clean cage and allow the wound to heal without wound dressing. Monitor the mouse for 3 days to ensure the mouse returns to normal activity.</li>
</ol>
2. Digit Collection and Tissue Preparation
<ol>
	<li>Euthanize the mouse using carbon dioxide (CO2) in a closed chamber, followed by cervical dislocation.</li>
	<li>Use a scalpel to sever the digit mid-way through the adjacent bone segment, the middle phalanx (P2) bone, at approximately the second ventral fat pad indent. Transfer the digit(s) to a 20 mL scintillation vial containing 10 mL fresh buffered zinc formalin fixative, an 18% formaldehyde solution (see Table of Materials). Fixative volume should be at least 20x the volume of the tissue present. 
	NOTE: Digits are collected at various timepoints after amputation to investigate the full regeneration response. Generally, for early blastema formation, bone histolysis, and wound closure the collection timepoints are 4&#8211;8 days post amputation (DPA). To investigate the early osteogenic blastema the collection timepoints are 9 and 10 DPA, and to visualize continued bone regeneration and mineralization, the collection timepoints are 14, 21, and 28 DPA. Unamputated digits are collected as well.</li>
	<li>Fix the digit for 24&#8211;48 h at room temperature with gentle mixing on a shaker.</li>
	<li>Remove the fixative and wash the digit 2x for 5 min in 5 mL of 1x phosphate buffered saline (PBS) at room temperature.</li>
	<li>Place 10 mL of fresh Decalcifier I (see Table of Materials), a 10% formic acid solution, in the scintillation vial and gently mix on a shaker for 2 h at room temperature. After 2 h, replace Decalcifier I with fresh solution and decalcify digit overnight at room temperature with gentle mixing on shaker.</li>
	<li>Remove the Decalcifier I and wash the digit 2x for 5 min in 5 mL of 1x PBS at room temperature.</li>
	<li>Process the digit through a graded ethanol series. This process can be performed manually in a 20 mL scintillation vial, using 10 mL of each liquid if less than 40 total digits.
	<ol>
		<li>Begin with 2 immersions in fresh 70% ethanol at room temperature with gentle mixing on a shaker, 1 h each, followed by 2 immersions in fresh 95% ethanol at room temperature with gentle mixing on a shaker, 1 h each.</li>
		<li>Complete the digit dehydration with 2 washes of fresh 100% ethanol at room temperature with gentle mixing on a shaker, 1 h each. The final 100% ethanol wash can be left overnight.</li>
	</ol>
	</li>
	<li>In the same scintillation vial, replace the 100% ethanol with 10 mL of fresh xylenes, and place the vial in a chemical fume hood for 1.5 h at room temperature. Repeat with a fresh wash of xylenes in a chemical fume hood for 1.5 h at room temperature for a total of 2 washes.</li>
	<li>Replace the xylenes with liquid paraffin wax melted to 68 &#176;C, and place the vial in an incubator at 68 &#176;C for 2 h, 2 times. After 4 total h of immersion in paraffin wax, embed the digit for paraffin sectioning. 
	NOTE: Digit processing for histology can be performed in an automated processor, following these same guidelines.</li>
	<li>Section digits at 4&#8211;5 &#181;m thickness using a microtome. Place sectioned samples on adhesive slides, using a 38&#8211;41 &#176;C water bath supplemented with a histological adhesive solution to ensure samples adhere to the slide. Place slides on a 37 &#176;C slide warmer to dry.</li>
</ol>
3. Immunohistochemical Staining of Adult Mouse Digits to Investigate Blastema Formation and Intramembranous Ossification
<ol>
	<li>Heat sectioned slides at 65 &#176;C for 45 min, followed by heating at 37 &#176;C for no less than 15 min to ensure samples adhere to the slide.</li>
	<li>Deparaffinize and rehydrate slides 2x with 5 min washes of xylenes, a graded ethanol series, and submersion in water. Place slides in a 50&#8211;100 mL capacity staining jar and keep immersed in 1x Tris Buffered Saline with Tween 20 (TBST). 
	NOTE: Do not allow samples to dry throughout the staining process. At any TBST step, slides can be incubated up to 2 h.</li>
	<li>Prepare a humidifying chamber. A 1-inch deep covered plastic slide box with moistened tissue paper at the base is sufficient.</li>
	<li>Prepare the heat retrieval solution for Runx2, Osterix (OSX), and Proliferating Cell Nuclear Antigen (PCNA). For the antigen retrieval of the early osteoprogenitor marker, Runx2, prepare a 1x solution of Tris-EDTA, pH 8. For the osteoblast marker, OSX, prepare a 1x solution of citrate buffer, pH 6. PCNA immunostaining can be performed in either solution.</li>
	<li>Prepare Proteinase K antigen retrieval solution for the blastema marker CXCR422 and the endothelial cell marker von Willebrand Factor 8 (vWF) by placing 100 &#956;L of Proteinase K solution per slide in a microcentrifuge tube and heating to 37 &#176;C (approximately 5 min). 
	NOTE: Antigen retrieval for Runx2, OSX, and PCNA is performed using heat retrieval, and CXCR4 and vWF antigen retrieval is performed via proteinase K treatment.</li>
	<li>For antibody retrieval requiring heat, immerse slides in a staining jar in the appropriate antigen retrieval solution. Heat slides to 95 &#176;C for 25 min, ensuring boiling does not occur. Remove staining jar from heat source and allow to cool at room temperature for 35 min.</li>
	<li>For Proteinase K antigen retrieval, lay slides flat in the humidifying chamber, and place 100 &#956;L of pre-warmed Proteinase K on the slide. Cover the slides with a small strip of parafilm to ensure the slides do not become dry. Incubate slides for 12 min at 37 &#176;C.</li>
	<li>Wash slides 3 times for 5 min in 1x TBST in staining jar after antigen retrieval.</li>
	<li>Remove slides from the staining jar and place in humidifying chamber. Place 6&#8211;7 drops of blocking solution (Table of Materials) on the slide, and cover the slide with fresh paraffin film to ensure the slide does not become dry. Incubate slides for 1 h at room temperature.</li>
	<li>Prepare the primary antibodies by diluting the antibodies into antibody diluent (Table of Materials). Vortex each primary antibody solution for 3 s. Primary antibodies derived from different host species may be combined. 
	NOTE: Immunohistochemical staining for Runx2 (rabbit anti-Runx2 antibody, 1:250 dilution, at a final concentration of 4 &#956;g/mL) combined with PCNA (monoclonal mouse anti-PCNA antibody, 1:2,000 dilution, at a final concentration of 0.5 &#956;g/mL), and OSX (rabbit anti-OSX antibody, 1:400 dilution, at a final concentration of 0.125 &#956;g/mL) combined with PCNA is shown in Figure 2. The mouse anti-PCNA antibody used in this study is highly specific and requires no additional blocking steps beyond those outlined in this protocol. Immunohistochemical staining using CXCR4 (rat anti-CXCR4 antibody, 1:500 dilution at a final concentration of 2 &#956;g/mL) and vWF (rabbit anti-human vWF VIII antibody, 1:800 dilution, at a final concentration of 41.25 &#956;g/mL) is shown in Figure 2. Primary antibodies were optimized and tested by our lab under the conditions in this protocol and are listed in the Table of Materials.</li>
	<li>Carefully remove the paraffin film, and gently drain the slide onto tissue paper to remove excess blocking solution. Place 100&#8211;200 &#956;L of primary antibody solution on the slide, replace parafilm cover, and return to the humidifying chamber. Incubate the slides in the closed humidifying chamber overnight at 4 &#176;C. 
	NOTE: Do not let the slides become dry while draining the blocking solution. This step is performed quickly.</li>
	<li>Carefully remove the parafilm, and gently drain the primary antibody solution from the slide. Place the slide in a staining jar containing fresh 1X TBST. Wash with fresh 1x TBST for 5 min 3 times.</li>
	<li>Prepare the secondary antibodies by combining with antibody diluent (Table of Materials). Vortex the secondary antibody solution for 3 s. Secondary antibodies conjugated to different fluorophores derived from different species may be combined. 
	NOTE: Fluorescent secondary antibodies are light sensitive. Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) for Runx2, OSX, and vWF, Alexa Fluor 568-conjugated goat anti-rat IgG (H+L) for CXCR4, and Alexa Fluor 647-conjugated goat anti-mouse IgG (H+L) for PCNA, diluted at 1:500 with a final concentration of 4 &#956;g/mL, were used in Figure 2.</li>
	<li>Place 200 &#956;L of secondary antibody solution on the slide, cover slide with fresh paraffin film, and return to humidifying chamber. Incubate the slides in the closed humidifying chamber for 45 min at room temperature. Ensure the humidifying chamber is closed to avoid light.</li>
	<li>Carefully remove the paraffin film and gently drain the secondary antibody solution from the slide. Place the slide in a staining jar containing fresh 1x TBST. Wash with fresh 1x TBST for 5 min 3x. Avoid light.</li>
	<li>Prepare nuclear stain. Add 20 &#956;L of DAPI (stock DAPI solution is 5 mg/mL in H2O) to 200 mL of 1x PBS and shake vigorously to ensure homogeneity. Immerse slides for 5 min in DAPI-PBS solution. Avoid light.</li>
	<li>Wash slides in dH2O for 3 min. Decant the water and allow the slides to air-dry or gently aspirate the water from the slide, ensuring that the samples are not disturbed while vacuuming. Avoid light.</li>
	<li>Carefully coverslip dry slides using 100 &#956;L of anti-fade mounting medium (Table of Materials); avoid the formation of air bubbles on the slide during the mounting step. Store the slides flat and allow the mounting medium to dry overnight at room temperature in a light-proof container. After the mounting medium is dry, store slides flat in a light-proof container in 4 &#176;C until ready to view. 
	NOTE: If unacceptable levels of air bubbles are present after mounting, the mounted slide can be gently placed in 1x PBS, the coverslip can be removed, and once the slide is rinsed again in water and re-dried, can be re-mounted.</li>
</ol>
4. Microscopy and Image Analysis
NOTE: Imaging and analysis using a fluorescence deconvolution microscope and associated software, equipped with 3 fluorescent filters (to visualize Alexa Fluor 488, 568, and 647 nm signals), plus DAPI (419 nm) is used in this experiment.
<ol>
	<li>Image the slide at 10x magnification to capture the entire blastema region. 
	NOTE: Proliferating osteoblast primary antibody detection utilizes secondary antibodies (Alexa Fluor 488 and 647) with non-overlapping emission spectra to minimize incorrect identification of co-labeled cells. Background subtraction of autofluorescent signal can be performed using the 568 nm filter to ensure integrity of the 488 or 647 signals. Blastema primary antibody detection utilizes secondary antibodies (Alexa Fluor 488 or 568). Background subtraction of autofluorescent signal can be performed with the 568 filter if using the 488-conjugated antibody, and 488 filter is using the 568-conjugated antibody. Autofluorescent signal is typically associated with the nail plate and erythrocytes throughout the digit, and is observed in the 488, 568, and 647 filters.</li>
</ol>
5. Sequential In Vivo Microcomputed Tomography (&#956;CT)
<ol>
	<li>Regenerating digits of the same animal scanned at 1 day prior to amputation (unamputated), 1 DPA, and at various time points until complete regeneration at 28 DPA using the vivaCT 40 (Table of Materials) is performed in this experiment and shown in Figure 3A.</li>
	<li>Outfit the &#956;CT with tubing to enable oxygen flow into and out of the &#956;CT animal chamber, ensuring that the out-flowing oxygen is equipped with an activated charcoal filter to trap the isoflurane. 
	NOTE: Ensure that the &#956;CT animal chamber is tightly enclosed; in this way, an animal nose-cone is not required for supplying isoflurane to the mouse during the scan.</li>
	<li>Prepare the &#956;CT scanning parameters. Digits are scanned at a voxel size of 10.5 &#956;m, at 55 kVp, 145 uA with 1000 projections per 180&#176; using continuous rotation, and with an integration time of 200 msec, resulting in a maximum of 213 slices per scan. Scans performed in this experiment are approximately 9 min long. A decreased electric current can be compensated with proportionally increased integration time but will result in longer scan times.</li>
	<li>Anesthetize the adult mouse using isoflurane; initially anesthetize at 3% in a chamber, followed by 1.5% isoflurane supplied to the &#181;CT animal chamber over the duration of the scan. Gently place the mouse in the &#181;CT animal chamber with the hindlimb digits arranged in close association, flat, ventral side-up with left paw on left side and right paw on right side on the scanning platform, followed by gently securing the digits in place using surgical tape. Apply ophthalmic ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Return the mouse to a clean cage and monitor the mouse until awake. Continue scanning the same mouse at bi-weekly to weekly time points to visualize the entire bone regeneration response.</li>
	<li>After scanning, allow up to several hours for &#956;CT image reconstruction to occur. Using the software provided with the &#956;CT, convert the files to a series of dicom files; each dicom file will correspond to one slice of the scan, therefore, if 213 slices have been scanned, 213 dicom files will be generated and uploaded to the company&#8217;s server.</li>
	<li>Download the dicom series in a single folder using a batch file downloader in a web browser.</li>
	<li>Download the BoneJ plugin23 and drag the file into the ImageJ plugin folder. In ImageJ, open the dicom file series as a stack by dragging and dropping the folder to the ImageJ bar. A 16-bit image stack displaying all the gray values will be opened. To display bone only, perform image thresholding (Image &#62; Adjust &#62; Threshold). 
	NOTE: When viewing ImageJ files, the ImageJ output will correspond to an inverted image of the digits; i.e., digits arranged in the scanning platform ventral side-up, with left paw on left side of platform and right paw on right side of platform, will appear as dorsal side-up, right paw on left side and left paw on right side of the ImageJ image stack.</li>
	<li>Once the threshold dialog box is opened, slide the bottom bar, corresponding to the upper threshold, to the far right, and adjust the top bar, corresponding to the lower threshold, until only the bone is highlighted red. The lower threshold value will be approximately 10,000&#8211;13,000 at a maximum signal intensity of 32,767. Click apply, and in the new dialog box click black background. Click OK.</li>
	<li>An 8-bit image displaying black and white values will be generated, with white corresponding to bone. Select the P3 bone by drawing a rectangle around it, and duplicate the stack. Generate a 3D image (Plugins &#62; 3D Viewer). To crop any unnecessary bone from the image, click the freehand selection tool, and circle the bone to be deleted. Right click, and select Fill Selection to delete bone. 
	NOTE: The steps described above apply to ImageJ 1.52h, Java 8 and may differ slightly when using a different version of ImageJ. For thresholding, we recommend the determination of an optimal value and using that value consistently.</li>
	<li>Quantify bone volume from the 3D rendering by using the BoneJ Volume Fraction Plugin (Plugins &#62; BoneJ &#62; Volume Fraction). A result window will appear, and bone volume (BV) will be displayed in mm3.</li>
	<li>Quantify bone length from the 3D rendering by using the ImageJ multipoint tool. 
	NOTE: Bone length is dynamic over the course of P3 regeneration; the length decreases between 7-10 DPA associated with osteoclast-mediated bone degradation11, and is followed by a period of distal bone regrowth between 14-28 DPA (Figure 3B). Length is measured from the central base of the P2/P3 joint to the farthest point of mineralization at the distal bone apex, but does not include degraded bone that has been completely detached (Figure 3B). BoneJ allows for the complete 3D assessment of the bone architecture; thus, the angle in which the bone is scanned will not alter the ability to measure length.</li>
	<li>Capture an image of the 3D rendering by clicking View, and take snapshot. Save image as a tif or jpeg file.</li>
</ol>

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The authors have nothing to disclose.

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><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&amp;amp;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...

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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 ...

adult-mouse-digit-amputation-regeneration-simple-model-to-investigate

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

8.7K Views.  Texas A&M University. P3 amputatie is een preklinisch model voor het onderzoeken van zoogdier epimorfe regeneratie en een krachtig instrument voor het identificeren van kritische cel populaties en mechanismen die endogene regeneratie controle. Het P3-model maakt het mogelijk om vroege en late blastemalcelpopulaties, de studie van revascularisatie tijdens regeneratie en het onderzoek naar intramembraneuze ossificatie zonder complexe vereisten voor botstabilisatie te identificeren. Het aantonen van de procedure met ...

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

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

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 ...

Medicine

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 ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

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 ...

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling structure that forms rapidly post-amputation and is required for limb regeneration. However, methods to study its precise function at the molecular level over the last decades have been limited due to a paucity of precise functional techniques and genomic information available in salamander model systems. Excitingly, the recent plethora of sequencing technologies coupled with the release of various salamander genomes and the advent of functional genetic testing methods, including CRISPR, makes it possible to re-visit these foundational experiments at unprecedented molecular resolution. Here, I describe how to perform the classically developed full skin flap (FSF) surgery in adult axolotls in order to inhibit wound epidermis formation immediately following amputation. The wound epidermis normally forms via distal migration of epithelial cells in the skin proximal to the amputation plane to seal off the wound from the outside environment. The surgery entails immediately suturing full thickness skin (which includes both epidermal and dermal layers) over the amputation plane to hinder epithelial cell migration and contact with the underlying damaged mesenchymal tissues. Successful surgeries result in the inhibition of blastema formation and limb regeneration. By combining this surgery method with contemporary downstream molecular and functional analyses, researchers can begin to uncover the molecular underpinnings of wound epidermis function and biology during limb regeneration.

This article describes how to perform a surgical method to inhibit wound epidermis formation during axolotl limb regeneration by immediately suturing full thickness skin over the amputation plane. This method allows researchers to investigate the functional roles of the wound epidermis during the early stages of limb regeneration.

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling ...

Biology

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 ...

Using Plate Fixation to Study Bone Healing and Limb Regeneration in Axolotls

Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged organs, including limbs, brain, heart, tail, and others. Studies on axolotl shed light on cellular and molecular pathways ruling progenitor activation and tissue restoration after injury. This knowledge can be applied to facilitate the healing of regeneration-incompetent injuries, such as bone non-union. In the current protocol, the femur osteotomy stabilization using an internal plate fixation system is described. The procedure was adapted for use in aquatic animals (axolotl, Ambystoma mexicanum). &#8805;20 cm snout-to-tail tip axolotls with fully ossified, mouse-size comparable femurs were used, and special attention was paid to the plate positioning and fixation, as well as to the postoperative care. This surgical technique allows for standardized and stabilized bone fixation and could be useful for direct comparison to axolotl limb regeneration and analogous studies of bone healing across amphibians and mammals.

Author Spotlight: Exploring Regeneration in Axolotls Through Insights in Cellular and Molecular Mechanisms, Bone Healing, and Implications for Human Therapies

A protocol for femoral osteotomy surgery with the use of internal plate fixation in mature axolotls is presented. The procedure can be used to perform comparative studies on limb regeneration and fracture healing in aquatic amphibians.

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged ...

Mechanical Characterization of Tissue Stiffness During Axolotl Limb Regeneration

Atomic Force Microscopy Measurements of Cartilage in Intact and Regenerating Axolotl Limbs

Mechanical forces provide important signals for normal cell function and pattern formation in developing tissues, and their role has been widely studied during embryogenesis and pathogenesis. Comparatively, little is known of these signals during animal regeneration. 
The axolotl is an important model organism for the study of regeneration, given its ability to fully restore many organs and tissues after injury, including missing cartilage and bone. Due to its crucial role as the main supporting tissue in the vertebrate body, regaining skeletal function during regeneration requires both the restoration of the missing structures as well as their mechanical properties. This protocol describes a method for processing axolotl limb samples for atomic force microscopy (AFM), which is the gold standard for probing cell and tissue mechanical properties at high spatial resolution. 
Taking advantage of the regenerative capabilities of the axolotl, this study measured the stiffness of limb cartilage during homeostasis and two stages of limb regeneration: tissue histolysis and cartilage condensation. We show that AFM is a valuable tool for gaining insights into dynamic tissue restructuring and the mechanical changes that occur during regeneration.

Author Spotlight: Exploring the Role of Mechanical Signals in Tissue Regeneration Through Atomic Force Microscopy 

In this protocol, we show how to prepare axolotl tissue for atomic force microscopy (AFM) and perform indentation measurements in intact and regenerating limb cartilage.

Mechanical forces provide important signals for normal cell function and pattern formation in developing tissues, and their role has been widely studied ...

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

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