This work was supported by a grant from the National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) 1R21AR079153 and a University of Connecticut Research Enhancement Program (REP) grant. The authors would like to acknowledge the assistance of Renata Rydzik from the University of Connecticut MicroCT Imaging Core facility.

Precise Growth Plate Injury Study Using Trilineage Fluorescent Mice

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The authors have no conflicts of interest to disclose.

The innovative use of tricolor collagen reporter mice enables the creation of growth plate defects with a predetermined size and location, significantly enhancing the accuracy of murine experimental models for growth plate injuries. Given the small size of the 2-week-old mice, it is critical to use a small 0.5 mm bur to create the injury to avoid weakening the limb and causing a full-thickness fracture. The surgeon must also apply just enough pressure when creating the defect to avoid drilling too deeply into the bone for the same reason. The use of the perioprobe is critical to confirming a consistent injury depth.
As with any surgery, it is important to confirm an adequate depth of anesthesia, confirmed by an occasional toe pinch and sterility is maintained throughout. Another surgical point of importance is that blunt dissection with a carver has been described because it avoids damaging soft tissue and helps to ensure the mice are able to ambulate immediately after recovery from anesthesia to reach the mother mouse for nutrition and comfort. In our experience, the wounds closed with sutures have remained successfully closed and wound clips are not required. Surgery on mice at 2 weeks of age is recommended to best mimic the young child that experiences growth plate fractures. One downside of this protocol is that given the unpredictable nature of birthing, the use of this mouse model requires the availability of the surgeon at short notice.
Regarding the positioning of the bur to create the defect, the protocol describes creating the injury using a mCherry/Texas red filter set that illuminates the hypertrophic zone within the growth plate because of the brightness of the collagen X fluorescence. To ensure the injury is created within the tibial growth plate, it is beneficial to slightly move the soft tissue opening to the left and right to confirm that the proximal tibial growth plate is in view, and not the femur. Switching between filter set channels to illuminate the proliferative chondrocyte zone or the adjacent bone sections is useful for confirming accurate placement relative to the location of the proliferative zone and adjacent bone sections.
While the proliferative chondrocyte zone and the epiphyseal and metaphyseal bone can be distinguished under fluorescence microscopy in the live mice, the real value of the Type II and Type I collagen reporters are realized during the histological analysis of the growth plate. Given the aqueous nature of cryo-histological processes, traditional chromogenic dye precipitation protocols are unsuitable due to the potential misalignment of color with fluorescent imaging caused by dehydration steps. Although the aqueous protocol yields staining patterns similar to those in paraffin sections, rapid post staining imaging is essential to prevent dye diffusion from the tissue. Utilizing 30% glycerol in distilled water as the mounting medium can decelerate this diffusion, allowing for multiple chromogenic staining on the same section, including cartilage with Safranin O/Fast Green.
The endochondral ossification process is clearly visible with red chondrocytes lining the evolving bony bridge (Figure 6). Additional use of immunohistochemistry techniques, for which there are many murine antibodies available, could further enhance mechanistic studies conducted in these transgenic mice. Altogether, the combination of faxitron, microCT, and cryo-histological imaging techniques in this transgenic mouse model offers a comprehensive understanding of macroscopic and microscopic changes that occur in response to growth plate injuries, paving the way for future therapeutic interventions to mitigate such adverse outcomes. Further genetic manipulations of these transgenic mice could be done to allow lineage tracing studies to understand the origin of the cells that are temporally and spatially involved in healing. Experimentation on mice with additional modifications would allow the study of cartilage diseases such as osteochondroma - an overgrowth of cartilage and bone near the growth plate.
The consistency of our model is demonstrated by the reproducible formation of bony bridges in all mice without needing to discard any mice from the group due to articular cartilage injury. This is an improvement over previous models that approached the growth plate from a cortical window below the growth plate and angled a sharp tool or bur upwards towards the growth plate and occasionally would overshoot into the articular cartilage. An additional injury of the articular cartilage does not mimic the commonly occurring growth plate injuries in children. The more precise injury of this animal model reduces the number of mice required per experiment and that is another improvement. The use of transgenic mice allows the researcher to focus the injury on sub-sections of the growth plate, such as the hypertrophic/provisionally calcified area or the epiphysis/resting zone/proliferative zone area, without affecting the articular cartilage. However, a limitation of this model is the variability in bony bridge volume, which can differ by up to 30% among injured animals. Consequently, detecting a clinically significant effect on bony bridge formation still necessitates a large number of animals to achieve statistical relevance.
Benefits to a mouse model as described here as compared to rat or rabbit growth plate injury models previously published7,9,10,14, include a lower number of animals used, cost reduction, an efficient replicate size due to reproducible bony bar formation, a shorter study time frame, and more precise injury placement due to live imaging of the triple transgenic mice. While not discussed in detail, this mouse model can be used to test tissue-engineered implants or biomaterials delivering growth factors. A notable limitation of this murine method is that the size of an implant used to deliver therapeutic drugs or cells is limited to the defect volume of roughly a 0.5 mm diameter sphere. Only larger animal models can accommodate the volume of test material that would be used in human patients. The bur defect created in this protocol is not the same geometry as a thin fracture and thus differs from actual human injuries. Nonetheless, the benefits of this mouse model are many, and the lateral approach avoids damaging the articular cartilage that would occur when approaching blindly above or below the growth plate in line with the tibial long axis. This methodology represents a substantial leap in growth plate injury research, providing a detailed and reproducible method for investigating pathology and evaluating new therapeutic strategies.

Bone growth plates play a pivotal role in the longitudinal growth of long bones during childhood and adolescence1. Situated at the ends of long bones, the growth plate comprises multiple zones, with chondrocytes being the key cellular components responsible for producing and maintaining this dynamic growth area. Endochondral ossification of the growth plate occurs to lengthen and expand the bones through a sequential progression of chondrocyte proliferation, hypertrophy, apoptosis, invasion by blood vessels, recruitment of osteoprogenitor cells, and finally, bone formation2. Since the growth plate is relatively softer than bone, it is highly susceptible to fracture when bones are overloaded during sports or other activities. The Salter-Harris classification outlines five distinct types of growth plate injuries3. The Type II fracture through the hypertrophic zone of the growth plate and the adjacent lower bone tissue is the most prevalent4. A bony bridge often forms in response to injuries of the hypertrophic zone or the adjacent bone and leads to premature fusion of the adjacent long bone sections5. Bony bridges impede the normal expansion of the growth plate. Currently, no preventative treatments are available for the bony bridge formation, and some are left untreated depending on the patient&#39;s age and the bony bridge size and location6. When limb malformation is severe, surgical options include removal followed by implanting interpositional materials like fat or silicone rubber or corrective osteotomy and bone lengthening procedures; yet a bony bridge may still reform6. More research is needed to prevent bony bridge formation and to improve the outcomes of children with bone growth plate injuries.
Several animal models have been established to explore the underlying mechanisms and develop new strategies to prevent bony bridge impairment of growth plates post injury7,8,9,10,11,12. These animal models frequently focus on the proximal tibial growth plate and distal femur growth plate as the primary injury site, given this is typically where human injuries occur. The animal bone defects are created either by a lateral approach similar to an actual fracture pathway or an approach from above or below the growth plate leading to a central drill hole in the growth plate. In a previously reported rat model, a growth plate defect is created by inserting a dental bur through a cortical window in the tibial midshaft and drilling upwards through the marrow towards the knee joint to centrally injure the growth plate7,13. Alternatively, a recent mouse model uses a lateral approach with a small-bore needle to create a planar needle track through the growth plate8. In a widely used rat model, the defect is created in the growth plate of the distal femur by drilling through the articular cartilage between the condyles9,14. In larger animals like rabbits and sheep, growth plate defects have been both laterally induced directly in the proximal tibia and the distal femur by drilling or cutting into the growth plate or by approaching from below and creating a central defect leaving the edges of the growth plate unaltered10,11,12,15.
Murine models for growth plate injuries are advantageous for mechanistic studies that can be accomplished with genetically modified mice, such as stem cell lineage tracing studies8. However, a significant challenge in murine or rat animal models is achieving consistent and precise injury to a particular sub-region of the growth plate. Injury of particular zones of the growth plate and adjacent bone is required to mimic one of the clinically relevant fracture paths described by the Salter-Harris classifications. The challenges to date in rodent models are primarily due to the lack of a visual means of identifying the substrata of the growth plate during the surgical creation of the injury. This protocol describes a refined technique for creating growth plate defects in targeted substrata of the murine growth plate by utilizing triple transgenic mice that express collagen I, II, and X fluorescent reporters16,17,18. The different colored fluorescence of these collagens in each of the primary zones of the growth plate allows visual discrimination of the various sections of the growth plate under a fluorescence stereo microscope during the surgical creation of the growth plate injury. The use of these transgenic mice allows for unprecedented injury accuracy in a young mouse at a comparable developmental stage to the children that are injured.

<table><tbody><tr><td>2-methyl-butane</td><td>Sigma Aldrich</td><td>M32631</td><td></td></tr><tr><td>Alcohol antiseptic pads</td><td>Acme United Corporation</td><td>H305-200</td><td></td></tr><tr><td>Axio Scan.Z1</td><td>Carl Zeiss AG</td><td>Axio Scan.Z1</td><td></td></tr><tr><td>AxioVision software</td><td>Carl Zeiss AG</td><td></td><td></td></tr><tr><td>Betadine solution (10% povidone-iodine)</td><td>Avrio Health L.P.</td><td>67618-150-01</td><td></td></tr><tr><td>Calcein</td><td>Sigma Aldrich</td><td>C0875</td><td></td></tr><tr><td>Calcein Blue</td><td>Sigma Aldrich</td><td>M1255</td><td></td></tr><tr><td>CFP filter set</td><td>Chroma Technology Corp.</td><td>49001</td><td></td></tr><tr><td>Cryomatrix</td><td>Thermo Scientific</td><td>6769006</td><td></td></tr><tr><td>Cryomolds</td><td>Fisher Scientific</td><td>Fisherbrand #22-363-554</td><td></td></tr><tr><td>Cryostat</td><td>Leica Biosystems</td><td>3050s</td><td></td></tr><tr><td>Cryostat blades</td><td>Thermo Scientific</td><td>3051835</td><td></td></tr><tr><td>Cryotape</td><td>Section Lab</td><td>Cryofilm 2C</td><td></td></tr><tr><td>Curved fine scissor&amp;nbsp;</td><td>Fine Science Tools</td><td>14061-11</td><td></td></tr><tr><td>Curved mosquito hemostatic forceps</td><td>HuFriedyGroup</td><td>H3</td><td></td></tr><tr><td>cy5 filter set</td><td>Chroma Technology Corp.</td><td>49009</td><td></td></tr><tr><td>DAPI</td><td>ThermoFisher Scientific</td><td>62247</td><td></td></tr><tr><td>DAPI filter set</td><td>Chroma Technology Corp.</td><td>49000</td><td></td></tr><tr><td>Dental bur (0.5 mm diameter)</td><td></td><td></td><td></td></tr><tr><td>Dental cleoid discoid carver</td><td>ACE Surgical Supply Inc.</td><td>6200097A-EA</td><td></td></tr><tr><td>Dry glass bead sterilizer (Inotech Steri 350)</td><td>Inotech Bioscience, LLC</td><td>IS-250</td><td></td></tr><tr><td>Ear punch</td><td>Fine Science Tools</td><td>24212-01</td><td></td></tr><tr><td>Electric heating pad</td><td></td><td></td><td></td></tr><tr><td>Electronic foot control</td><td>Nouvag AG</td><td>1866nou</td><td></td></tr><tr><td>Electronic motors 31 ESS</td><td>Nouvag AG</td><td>2063nou</td><td></td></tr><tr><td>Environmental surface barrier (3 x 12 inch tube sox)</td><td>Patterson Companies, Inc.</td><td>BB-0312H</td><td></td></tr><tr><td>Ethanol (70%)</td><td></td><td></td><td></td></tr><tr><td>Ethiqa XR (buprenorphine extended-release injectable suspension) 1.3 mg/mL</td><td>Fidelis Animal Health</td><td>86084-100-30</td><td></td></tr><tr><td>Faxitron x-ray cabinet</td><td>Kubtech Scientific</td><td>Parameter</td><td></td></tr><tr><td>Fluorescence Stereomicroscope</td><td>Carl Zeiss AG</td><td>Lumar V12</td><td></td></tr><tr><td>GFP filter set</td><td>Chroma Technology Corp.</td><td>49020</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Sigma Aldrich</td><td>ARK2183</td><td></td></tr><tr><td>Glass microscope slides</td><td>Thermo Scientific</td><td>3051</td><td></td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G5516</td><td></td></tr><tr><td>Graefe forceps</td><td>Fine Science Tools</td><td>11051-10</td><td></td></tr><tr><td>Handpiece (contra angle 32:1 push button)</td><td>Nouvag AG</td><td>5201</td><td></td></tr><tr><td>Implantology/oral surgery system control unit (Straumann)</td><td>Nouvag AG</td><td>SEM&amp;nbsp;</td><td></td></tr><tr><td>Instant sealing sterilization pouch with dual internal/external process indicators (3 1/2 x 5 1/4&amp;nbsp; inch)</td><td>Fisher Scientific</td><td>01-812-50</td><td></td></tr><tr><td>Instant sealing sterilization pouch with dual internal/external process indicators (5 4/1 x 10 inch)</td><td>Fisher Scientific</td><td>01-812-54</td><td></td></tr><tr><td>Insulin syringe (29 G)</td><td>Exel International</td><td>26028</td><td></td></tr><tr><td>Isoflurane&amp;nbsp;</td><td>Dechra Pharmaceuticals plc</td><td>17033-091-25</td><td></td></tr><tr><td>Isoflurane anesthetic system</td><td></td><td></td><td></td></tr><tr><td>mCherry filter set</td><td>Chroma Technology Corp.</td><td>39010</td><td></td></tr><tr><td>Micro-dissecting scissor</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>S5761</td><td></td></tr><tr><td>Needle (20 G)</td><td>Becton, Dickinson and Company</td><td>305178</td><td></td></tr><tr><td>Needle holder</td><td>HuFriedyGroup</td><td>NHCW</td><td></td></tr><tr><td>Neutral buffered formalin (10%)</td><td>Sigma Aldrich</td><td>HT501128-4L</td><td></td></tr><tr><td>Non-sterile applicator swabs</td><td>Allegro Industries</td><td>205</td><td></td></tr><tr><td>Non-woven gauze (3 x 3 inch)</td><td>Fisher Scientific</td><td>22028560</td><td></td></tr><tr><td>Norland Optical Adhesive, 61</td><td>Norland Optical</td><td>Norland Optical Adhesive, 61</td><td></td></tr><tr><td>Ophthalmic ointment (Optixcare eye lube)</td><td>CLC Medica</td><td></td><td></td></tr><tr><td>PBS</td><td>Sigma Aldrich</td><td>P5368</td><td></td></tr><tr><td>Periodontal probe</td><td>HuFriedyGroup</td><td>PQW</td><td></td></tr><tr><td>Phosphate buffered saline (PBS) pH 7.4 (1x)</td><td>Gibco, by Life Technologies</td><td>10-010-023</td><td></td></tr><tr><td>Plastic microscope slides</td><td>Electron Microscopy Sciences</td><td>71890-01</td><td></td></tr><tr><td>Professional clipper/trimmer (Wahl Classic Peanut)</td><td>Wahl Clipper Corporation</td><td>8685</td><td></td></tr><tr><td>Roller</td><td>Electron Microscopy Sciences</td><td>62800-46</td><td></td></tr><tr><td>Scanco Medical software</td><td>SCANCO Medical</td><td>Scanco &amp;mu;CT 50</td><td></td></tr><tr><td>Sodium acetate anhydrous</td><td>Sigma Aldrich</td><td>S2889</td><td></td></tr><tr><td>Sodium nitrite</td><td>Sigma Aldrich</td><td>S2252</td><td></td></tr><tr><td>Sodium tartrate dibasic dihydrate</td><td>Sigma Aldrich</td><td>T6521</td><td></td></tr><tr><td>Specimen disc</td><td>Leica Biosystems</td><td>14037008587</td><td></td></tr><tr><td>Stainless steel #15 surgical blade&amp;nbsp;</td><td>Aspen Surgical Products, Inc.</td><td>371615</td><td></td></tr><tr><td>Sterile surgical gloves</td><td>Cardinal Health, Inc.</td><td>2D72PT65X</td><td></td></tr><tr><td>Sterile towel drape (18 x 26 inch)</td><td>IMCO</td><td>4410-IMC</td><td></td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S9378</td><td></td></tr><tr><td>Syringe (1 mL)</td><td>Becton, Dickinson and Company</td><td>309659</td><td></td></tr><tr><td>Undyed braided coated vicryl suture (5-0)</td><td>Ethicon Inc.</td><td>J490G</td><td></td></tr><tr><td>UV black light</td><td>General Electric</td><td>F15T8-BLB</td><td></td></tr></tbody></table>

tricolor transgenic murine model for studying growth plate injury

The cartilage growth plates in the bones of children enable limb lengthening but are weak relative to the bone, making them prone to fracturing when bones are overloaded.&#160;Better treatments for severely fractured growth plates are needed because the response to injury is a bony bridge that prematurely fuses the growth plate, leading to stunted and/or crooked limbs. Murine models of growth plate injury are advantageous for mechanistic studies, but&#160;are challenging because it is difficult to visualize and precisely injure the small growth plates in young mice.&#160;We describe here an improved growth plate injury model using transgenic mice with tri-lineage fluorescent reporters for collagen types I, II, and X.
These mice show native fluorescence associated with the three primary substrata of the growth plate. A growth plate injury similar to a Salter-Harris Type II injury is created reproducibly with a bur using the hypertrophic section of the growth plate as a reference during live imaging under fluorescence stereo microscopy guidance. Frozen histology analysis of the native fluorescence simplifies assessing the cellular response to injury. This methodology represents a substantial leap in growth plate injury research, providing a detailed and reproducible method for investigating pathology and evaluating new therapeutic strategies.

This protocol utilizes trilineage fluorescent reporter mice to induce a lateral growth plate defect in the proximal tibia with precision by leveraging the inherent red fluorescence emitted by type X collagen for surgical guidance. The view the surgeon has while looking through the stereomicroscope eyepiece with the mCherry filter set is shown in Figure 2D. The native Type X fluorescence allows the surgeon to place the bur in the hypertrophic zone and create an injury that mimics a common type of growth plate injury leading to a bony bridge (Figure 2F). The fluorescence under the red channel is the brightest and, therefore, recommended for use during bur placement. Alternatively, defect creation could be guided by using other colors of the native fluorescence of the triple transgenic mice if the goal of the experiment is to study injuries to other zones of the growth plate than the hypertrophic zone and adjacent calcified region.
The creation of a Salter-Harris Type II-like defect in the hypertrophic zone of the growth plate and the adjacent lower bone tissue, using a 0.5 mm diameter dental bur, was validated through microCT and cryo-histology imaging of the injured (time 0) proximal tibias compared to the uninjured lateral controls in N = 3 mice (Figure 4). The defects were difficult to see in the 3D microCT images but were detectable in the 2-D cross sections (Figure 3A,B,E,F). Figure 3G displays the distribution of type I collagen-producing bone cells (green fluorescence), type II collagen-producing proliferative chondrocytes (cyan fluorescence), and type X collagen-producing hypertrophic chondrocytes. In the image of the injured mouse (Figure 4G), there is a disruption of the hypertrophic zone, the provisionally calcified layer, and some of the newest formed bone relative to the control with the proliferative zone only slightly disturbed. Safranin O/Fast Green staining (Figure 4H) best illustrates the location of the defect within the injured growth plate since all cells are clearly visible.
X-ray analysis provides some insight into live mice as to the impact of this type of growth plate injury on tibia length and bony bridge formation over time (Figure 3). Comparative imaging between uninjured (Figure 3A) and injured (Figure 3B) tibiae, taken before surgery and 3 weeks post surgery, reveals a large amount of limb growth, thinning of the growth plates, and a distinct opaque region that has developed in the injured growth plate area at 3 weeks. This opacity within the growth plate is not present in the uninjured counterpart nor the mice before surgery. Faxitron is thus one way of observing pathological changes induced by the injury in live mice, such as the formation of a bony bridge and changes in limb length.
MicroCT imaging of dissected bones offers a detailed visualization of bony bridge formation within the injured growth plates three weeks after surgery (Figure 5). As seen in the images from six different injured mice shown in Figure 5, there is consistent bony bridge development in all mice. Utilizing Scanco Medical software, the bony bridge volume was calculated by reviewing each section of the proximal tibial growth plate, delineating the area of the bony bridge (Figure 5B) with the select tool, and then, integrating each section area throughout the entire growth plate volume to get the total volume24. The bony bridge volume calculated this way was 0.0761 mm3 &#177; 0.0246 (mean &#177; standard deviation, N = 6). The majority of the bony bridges form near the middle of the growth plate despite the lateral approach, which injures the outer edge as well as the center of the growth plate. This phenomenon can be attributed to the fact that mesenchymal stem cells (MSCs) from the bone marrow, rather than the perichondrium, are responsible for bony bridge formation25.
In these tricolor transgenic mice, cryo-histological analysis of the injured growth plate is enriched by the native collagen fluorescence (Figure 6). It reveals the complex interplay of bone cells and chondrocytes at the injury site. MicroCT images shown in Figure 6J,K were provided to the histology technician to guide the embedding and sectioning. The type I collagen-producing bone cells are seen in Figure 6L,O,P (green fluorescence), while type II collagen-producing proliferative chondrocytes are seen in Figure 6L,O,Q (cyan fluorescence). Type X collagen-producing hypertrophic chondrocytes are seen in Figure 6L,O,R (red fluorescence). This multicolor fluorescence approach enables a detailed examination of postsurgery chondrocyte differentiation within the bony bridge area against a backdrop of mineralized tissue. DAPI staining was used to confirm the distribution of all cell types within the growth plate area (Figure 6M). The Safranin O/Fast Green staining demonstrates the composite and structural organization of cartilage and bone within the injured growth plate (Figure 6N). Imaging these stained sections under a Cy5 filter set notably brightens the resting zone cells at the interface between the epiphyseal bone and cartilage.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66841/66841fig03.jpg" /> 
Figure 3: X-ray images of contralateral control and injured mouse tibiae. (A) X-ray images of the contralateral control tibia are taken just before injury when mice are 2 weeks old and at 3 weeks after surgery when the mice are 5 weeks old, demonstrating the extent of growth that occurs during this period. (B) Injured tibia from the same mouse at the same time points as in (A). The landmarks used for tibia length measurements are the apex of the proximal tibia head to the end of the tibia at the ankle joint (red double-headed arrows). The opaque bony bridge is visible in the injured proximal tibia growth plate at 5 weeks. Scale bars = 5.00 mm. <a href="https://www.jove.com/files/ftp_upload/66841/66841fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66841/66841fig04.jpg" /> 
Figure 4: MicroCT and histological images of time zero contralateral control and injured mouse proximal tibiae. (A,E) and (B,F) depict 3D and transverse 2D microCT views, with the defect indicated by red arrows in (E) and (F). (C,G) Composite merged cryo-histological images merging three innate fluorescence layers with a mineralized tissue layer. Green cells (Col3.6GFPtpz) are the type I collagen-producing bone cells, cyan blue colored cells (Col2A1GFPcyan) are type II collagen-producing proliferative chondrocytes, and red cells (Col10A1RFPchry) are type X collagen-producing hypertrophic chondrocytes. (D,H) Safranin O/Fast Green staining of the same region as (C) and (G). Scale bars = 1.0 mm. <a href="https://www.jove.com/files/ftp_upload/66841/66841fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66841/66841fig05.jpg" /> 
Figure 5: MicroCT images of bony bridges formed by this protocol. (A,C,E,G,I,K) Transverse cross-sections of the proximal tibial growth plate of six different mice at 3 weeks after bur defect creation. Bony bridge outlined by a red dotted line in (A). (B,D,F,H,J,L)&#160;3D reconstructions with a longitudinal plane cut away. Bony bridge outlined by a red dotted line in (B). Scale bars = 1.0 mm. <a href="https://www.jove.com/files/ftp_upload/66841/66841fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66841/66841fig06.jpg" /> 
Figure 6: MicroCT and histological images of contralateral control and injured mouse proximal tibia with bony bridge formation. (A,J) and (B,K) depict 3D and transverse 2D microCT views, with the bony bridge indicated by yellow arrows in (J) and (K). (C,L) Composite merged cryo-histological images merging three innate fluorescence layers with a mineralized tissue layer. Green cells (Col3.6GFPtpz) are the type I collagen-producing bone cells, cyan blue colored cells (Col2A1GFPcyan) are type II collagen-producing proliferative chondrocytes, and red cells (Col10A1RFPchry) are type X collagen-producing hypertrophic chondrocytes. The white box indicates the higher magnification shown in panels F and O. (D,M) The mineralized tissue and DAPI staining in the growth plate area of panels C and L. (E,N) Safranin O/Fast Green staining of the same region as (D) and (M) scanned with cy5 fluorescence. (F,O) A higher magnification of the growth plate area in the merged image of panels C and L. (G-I,P-R) Individual channels of the native fluorescence shown with a mineralized tissue backdrop. Scale bars = 1.0 mm (A-E) and (J-N), = 250 &#181;m in (F-I) and (O-R). <a href="https://www.jove.com/files/ftp_upload/66841/66841fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Tricolor Transgenic Murine Model for Studying Growth Plate Injury

This protocol describes an improved mouse model for adolescent bone growth plate injuries. Using transgenic mice with tri-lineage fluorescent reporters for collagen types I, II, and X, the primary matrices associated with three different substrata of the growth plate, injury placement is guided by native fluorescence under the microscope.

The cartilage growth plates in the bones of children enable limb lengthening but are weak relative to the bone, making them prone to fracturing when bones ...

tricolor-transgenic-murine-model-for-studying-growth-plate-injury

Research

JoVE Journal

Medicine

556 Views.  University of Connecticut Health Center. Ik wil een betere behandeling ontwikkelen voor kinderen met botbreuken. Concreet wil ik botvorming in de gebroken kraakbeengroeischijf voorkomen, omdat anders. Het gebroken ledemaat zal normaal gesproken niet langer worden.Stap één, we moesten een nauwkeuriger muismodel ontwikkelen om nieuwe therapieën te testen en het werkingsmechanisme te bepalen. Conventionele behandelingen omvatten het wegsnijden van de benige brug na een verwonding en het inbre...

Video: Auteur in de schijnwerpers: voortschrijdend model voor groeischijfletsel met behulp van trilineage fluorescerende reporters bij muizen

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

Cancer Research

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 ...

Developmental Biology

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

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

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

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

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

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Bioengineering

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 ...

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found at the end of all long bones in children that is responsible for longitudinal bone growth. Once damaged, cartilage tissue within the growth plate can undergo premature ossification and lead to unwanted bony repair tissue, which forms a "bony bar." In some cases, this bony bar can result in bone growth deformities, such as angular deformities, or it can completely halt longitudinal bone growth. There is currently no clinical treatment that can fully repair an injured growth plate. Using an animal model of growth plate injury to better understand the mechanisms underlying bony bar formation and to identify ways to inhibit it is a great opportunity to develop better treatments for growth plate injuries. This protocol describes how to disrupt the rat proximal tibial growth plate using a drill-hole defect. This small animal model reliably produces a bony bar and can result in growth deformities similar to those seen in children. This model allows for investigation into the molecular mechanisms of bony bar formation and serves as a means to test potential treatment options for growth plate injuries.

The growth plate is a cartilaginous region in children's long bones where longitudinal growth occurs. When injured, bony tissue can form and impair growth. We describe a rat model of growth plate injury that leads to bony repair tissue, allowing the study of repair mechanisms and growth plate regeneration strategies.

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found ...

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 ...

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms and pharmaceutical treatments of osteoporosis. Cathepsin K (Ctsk)-Cre mice have been widely used for functional studies of osteoclasts. The signal transducer and activator of transcription 3 (STAT3) is relevant in bone homeostasis, but its role in osteoclasts in vivo remains poorly defined. To provide the in vivo evidence that STAT3 participates in osteoclast differentiation and bone metabolism, we generated an osteoclast-specific Stat3 deletion mouse model (Stat3 fl/fl; Ctsk-Cre) and analyzed its skeletal phenotype. Micro-CT scanning and 3D reconstruction implied increased bone mass in the conditional knockout mice. H&#38;E staining, calcein and alizarin red double staining, and tartrate-resistant acid phosphatase (TRAP) staining were performed to detect bone metabolism. In short, this protocol describes some canonical methods and techniques to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

This protocol describes a canonical method to understand the critical genes controlling osteoclast activity in vivo. This method uses a transgenic mouse model and some canonical techniques to analyze skeletal phenotype.

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms ...

Tricolor Transgenic Murine Model for Studying Growth Plate Injury

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