We thank Amanda Fair, the CMI, and Kathy Sweet and the ORL at UM for assistance with &#181;CT imaging and analysis. Funding: BL Funded by 1K08GM109105-01 and Plastic Surgery Foundation National Endowment Award.

הצהרת אתיקה: נהלי כל החיה בוצעו בהתאם קפדן עם תרגול חיה טוב כפי שהוגדר בהנחיות המופיעות במדריך לשימוש וטיפול בחי מעבדה: המהדורה השמינית של המכון לחקר בעלי החיים מעבדה (ILAR, 2011) והיו אושר על ידי הוועדה המוסדית הטיפול בבעלי חיים ושימוש באוניברסיטת מישיגן (PRO0001553). 1. ניתוחי עכבר הערה: C57BL הישן 8-10 השבוע השתמש / 6 עכברים. גם בגילים אחרים, רקעים, וזנים של עכברים יכולים לשמש כדי לבדוק תנאים שונים או איפור גנטי. עבור חלק tenotomy של ההליך, לשמור על תנאים סטריליים באמצעות מסיכת פנים, כובע שיער, ושמלת סטרילי, כפפות, ומכשירים. אתרי כירורגי צריכים להיות מוכנים סטרילי עם povidone- יוד וכורך סטרילי. הימנע היפותרמיה באמצעות כרית חימום וטרינרים והתחממות פתרונות החייאה עד 37 מעלות צלזיוס לפני ניהול כלליtion. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים את העכבר באמצעות 2.5% isoflurane בשאיפה. השתמש חרטום לעזוב את dorsum ואחורי-גפיים נגישים בקלות. התאם את שיעור ממשל isoflurane לשמור על קצב מתאים נשימה וצבע של קרומים ריריים ולהבטיח הרדמה על ידי בדיקת טונוס שרירים, קמצוץ הבוהן, ורפלקס מצמוץ. החל משחת עיניים תפל לעיניו של העכבר כדי למנוע פגיעה בעין יבשה במהלך ההליך. לשמור על טמפרטורת גוף מתאימה באמצעות כרית חימום וטרינרים או circulator מים חם מתחת לשדה הניתוח. </li><li style=";text-align:right;direction:rtl"> לנהל עצירות, 0.1 מ&quot;ג / קילוגרם, תת עורי מייד לפני הניתוח לשיכוך כאבים. </li><li style=";text-align:right;direction:rtl"> מקרוב לגלח את האתר כירורגית על הכף האחורית השמאלית באמצעות קוצץ מהעקב עד הברך. </li><li style=";text-align:right;direction:rtl"> מקרוב לגלח dorsum של העכבר, מתחיל מעל עמוד השדרה והארכת האזור המגולח בצד השמאל של קו האמצע הגב, ניקוי שטח לפחות 2 סנטימטרים X 3 סנטימטר כדי להתאים את פציעת הכווייה. </li><li style=";text-align:right;direction:rtl">הכן את האתר כירורגית, כולל הכף האחורית בברך באמצעות סקראבס povidone- יוד 3 לסירוגין. </li><li style=";text-align:right;direction:rtl"> לבצע חתך אורכי לאורך ההיבט המדיאלי של גיד אכילס עזב. להאריך את החתך כך ניתן דמיין בקלות גיד אכילס; כ 0.5 סנטימטר. </li><li style=";text-align:right;direction:rtl"> בצע tenotomy אכילס עם נתיחה חדה של הגיד באמצע עם מספריים חדים רקמה. הכנס להב אחד המספריים הרקמה במטוס הרקמה מתחת לגיד ולנתח יחד את המטוס עד שהלהב הוא באמצע הגיד. סגור את להבי המספריים לנתק את הגיד בצורה חדה. הערה: ודא הכללה של כל הגידים האחוריים כוללים הגסטרוקנמיוס, soleus, וגידי Plantaris. </li><li style=";text-align:right;direction:rtl"> להשיג עצירת דימום על ידי הפעלת לחץ עם גזה סטרילית (לא אמור להיות דימום מינימאלי) ולסגור את החתך בעור עם תפר 5-0 vicryl. </li><li style=";text-align:right;direction:rtl"> לבצע צריבת העובי החלקית הגב עם בלוק אלומיניום במשקל גרם 35 עם measuremen המשוערTS 2 סנטימטרים X 2 ס&quot;מ על 3 סנטימטר מחומם ל -60 מעלות צלזיוס באמבט מים להחיל את dorsum המגולח של העכבר במשך 17 שניות. הערה: השג את עומק הכווייה המתאים על ידי מנוחה הבלוק על גבי העכבר הרדים, להבטיח כי את כל פני השטח של הבלוק הוא במגע עם העכבר, לעומת זאת להימנע מהחלה כל לחץ נוסף לבלוק, כלומר, לאפשר את כוח משיכה כדי להיות הכוח היחיד שהחזיק את הגוש במקום. ידית פלסטיק דקיקה שצמודה אל צד אחד של הבלוק היא נוחה למניפולציה, ייצוב, ואחזור מאמבט המים החם. זו תיצור שטח גוף הכולל של כ -30% לשרוף בC57BL / 6 עכבר 8-10 בן שבוע. כווייה קשר זה נבחרה על פני שיטות אחרות (צריבת להבה או לחלוט) בשל האחידות של עומק כווייה על הפצע והשחזור בין בעלי החיים. </li><li style=";text-align:right;direction:rtl"> ייבש את אתר הכווייה בגזה ולהחיל הלבשה Tegaderm. </li><li style=";text-align:right;direction:rtl"> לנהל נוזלי החייאה חיממו: פתרון מיליליטר 1 של Lactated רינגרזריקת intraperitoneal ו -0.5 הזרקה תת עורית מיליליטר. ממשל חד פעמי זה של החייאה הוא נאות להתאוששות מהפציעה הכווייה וtenotomy. הערה: אל תחזור עכברים מורדמים לכלובי דיור עם עכברים אחרים, בית בנפרד בכלובים נקיים תחת ניטור עד עכברים הם התאוששו באופן מלא. התאוששות אופיינית מתרחשת בתוך 1-6 שעות. אתרי צריבה בדרך כלל לרפא בתוך 2-4 שבועות ומסובכים לעתים רחוקות על ידי זיהום פצע אם המשיכו בסביבות דיור נקיות. </li><li style=";text-align:right;direction:rtl"> לנהל 0.1 מ&quot;ג / קילוגרם עצירות הזרקה תת עורית בכל 12 שעות במשך 3 ימים לאחר ההליך. צג צמיחת HO עם סריקות μCT סידוריים במרווחים מתאימים. צמיחת עצם מחוץ לרחם היא ראשונה שלאחר ניתוח בסביבות 3 שבועות ניכרו על ידי μCT. הערה: רוב פיתוח HO הושלם על ידי 9 שבועות לאחר ניתוח. אנחנו לא נצפו הבדל בנפח HO או מיקום עם μCT חזר סריקות דו שבועי במשך 15 שבועות בהשוואה לנקודת סיום אחת μCT לסרוק בגיל 15 שבועות.בסופו של הניסויים, להרדים את כל העכברים עם CO 2 משאיפת פי הנחיות מוסדיות ולאמת מוות עם נקע בצוואר הרחם לאחר 10 דקות. </li></ol> 2. μCT רכישה וניתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אבטח את העכבר מורדם על מיטת הסורק במצב שכיבה. הדבק את הגפיים האחוריות היטב למיטה כדי למנוע נשימת חפץ תנועה. כלול את האוויר, מים, וhydroxyapatite מכיל פנטום מתחת לעכבר לכיול תמונה. </li><li style=";text-align:right;direction:rtl"> פתח את תוכנת עצם ניתוח ולהגדיר אזור של העניין (ROI) המקיף את שני גפיים אחוריות ממפרק ירך proximally לקצה הכף האחורית distally. להשיג תמונה באמצעות הפרמטרים הבאים:. 80 קילו וולט, 500 מיקרו-אמפר ו1,300 חשיפה msec, גודל voxel 48 מיקרומטר 14,15 </li><li style=";text-align:right;direction:rtl"> לכייל את התמונה ליחידות Hounsfield (האוניברסיטה העברית) על ידי ציור את ההחזר על ההשקעה בכל אחת משלושת תאי הפנטום ומזין את הצפיפות הממוצעת בשדות המתאימים בהתוכנה. </li><li style=";text-align:right;direction:rtl"> שימוש באפשרות &quot;חוטף&quot; בתוכנה, רי-אוריינט את התמונה כך השוקה של הגפיים האחורי השמאלי מקבילה לאורך ציר Z כדי לאפשר את התצוגה אנטומיים הברורה לתיחום של מבני orthotopic קליפת המוח עצם וHO. </li><li style=";text-align:right;direction:rtl"> החל בברך, לגלול distally דרך פרוסות התמונה עד HO הוא נתקל. שימוש בכלי שגם במדריך, לעשות את ההחזר על ההשקעה סביב עצם חוץ רחמי על כל פרוסה -5 ממשיכה distally דרך הכפה או עד HO כבר עלה. השתמש בכלי להסיק להאריך ולתפור ROIs יחד להחזר על השקעה אחד שמכיל את כל HO. </li><li style=";text-align:right;direction:rtl"> הפוך את ההחזר על השקעת 3D ולבחור את תפריט הניתוח. חשב את נפח העצם על ידי הגדרת ערכי הסף התחתונים ועליונים בצורה הטובה ביותר להציג את חלון העצם. השתמש באותם ערכי סף שנקבעו לכל הסריקות. </li></ol>

<ol>
	<li>Leblanc, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP-9-induced+muscle+heterotopic+ossification+requires+changes+to+the+skeletal+muscle+microenvironment.">BMP-9-induced muscle heterotopic ossification requires changes to the skeletal muscle microenvironment.</a> J Bone Miner Res. 26 (6), 1166-1177 (2011).</li><li>Shore, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoinductive+signals+and+heterotopic+ossification.">Osteoinductive signals and heterotopic ossification.</a> J Bone Miner Res. 26 (6), 1163-1165 (2011).</li><li>Wosczyna, M. N., Biswas, A. A., Cogswell, C. A., Goldhamer, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+progenitors+resident+in+the+skeletal+muscle+interstitium+exhibit+robust+BMP-dependent+osteogenic+activity+and+mediate+heterotopic+ossification.">Multipotent progenitors resident in the skeletal muscle interstitium exhibit robust BMP-dependent osteogenic activity and mediate heterotopic ossification.</a> J Bone Miner Res. 27 (5), 1004-1017 (2012).</li><li>Potter, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+ossification+following+combat-related+trauma.">Heterotopic ossification following combat-related trauma.</a> J Bone Joint Surg Am. 92, 74-89 (2010).</li><li>Van den Bossche, L., Vanderstraeten, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+ossification:+a+review.">Heterotopic ossification: a review.</a> J Rehabil Med. 37 (3), 129-136 (2005).</li><li>Chakkalakal, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Acvr1+R206H+knock-in+mouse+has+fibrodysplasia+ossificans+progressiva.">An Acvr1 R206H knock-in mouse has fibrodysplasia ossificans progressiva.</a> J Bone Miner Res. 27 (8), 1746-1756 (2012).</li><li>Yu, P. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+type+I+receptor+inhibition+reduces+heterotopic+[corrected]+ossification.">BMP type I receptor inhibition reduces heterotopic [corrected] ossification.</a> Nat Med. 14 (12), 1363-1369 (2008).</li><li>Culbert, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alk2+regulates+early+chondrogenic+fate+in+fibrodysplasia+ossificans+progressiva+heterotopic+endochondral+ossification.">Alk2 regulates early chondrogenic fate in fibrodysplasia ossificans progressiva heterotopic endochondral ossification.</a> Stem Cells. 32 (5), 1289-1300 (2014).</li><li>Dinther, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ALK2+R206H+mutation+linked+to+fibrodysplasia+ossificans+progressiva+confers+constitutive+activity+to+the+BMP+type+I+receptor+and+sensitizes+mesenchymal+cells+to+BMP-induced+osteoblast+differentiation+and+bone+formation.">ALK2 R206H mutation linked to fibrodysplasia ossificans progressiva confers constitutive activity to the BMP type I receptor and sensitizes mesenchymal cells to BMP-induced osteoblast differentiation and bone formation.</a> J Bone Miner Res. 25 (6), 1208-1215 (1359).</li><li>Peterson, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burn+injury+enhances+bone+formation+in+heterotopic+ossification+model.">Burn injury enhances bone formation in heterotopic ossification model.</a> Ann Surg. 259 (5), 993-998 (2014).</li><li>Scott, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+review+of+models+of+ectopic+bone+formation.">Brief review of models of ectopic bone formation.</a> Stem Cells Dev. 21 (5), 655-667 (2012).</li><li>Tannous, O., Griffith, C., O'Toole, R. V., Pellegrini, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+ossification+after+extremity+blast+amputation+in+a+Sprague-Dawley+rat+animal+model.">Heterotopic ossification after extremity blast amputation in a Sprague-Dawley rat animal model.</a> J Orthop Trauma. 25 (8), 506-510 (2011).</li><li>Tannous, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+bone+formation+about+the+hip+undergoes+endochondral+ossification:+a+rabbit+model.">Heterotopic bone formation about the hip undergoes endochondral ossification: a rabbit model.</a> Clin Orthop Relat Res. 471 (5), 1584-1592 (2013).</li><li>Peterson, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+heterotopic+ossification+through+remote+ATP+hydrolysis.">Treatment of heterotopic ossification through remote ATP hydrolysis.</a> Sci Transl Med. 6 (255), 255ra132 (2014).</li><li>Peterson, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+of+burn+induced+heterotopic+ossification+using+transcutaneous+Raman+spectroscopy.">Early detection of burn induced heterotopic ossification using transcutaneous Raman spectroscopy.</a> Bone. 54 (1), 28-34 (2013).</li><li>Perosky, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+of+heterotopic+ossification+using+near-infrared+optical+imaging+reveals+dynamic+turnover+and+progression+of+mineralization+following+Achilles+tenotomy+and+burn+injury.">Early detection of heterotopic ossification using near-infrared optical imaging reveals dynamic turnover and progression of mineralization following Achilles tenotomy and burn injury.</a> J Orthop Res. 32 (11), 1416-1423 (2014).</li><li>Peterson, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Aging+on+Osteogenic+Response+and+Heterotopic+Ossification+Following+Burn+Injury+in+Mice.">Effects of Aging on Osteogenic Response and Heterotopic Ossification Following Burn Injury in Mice.</a> Stem Cells Dev. , (2014).</li><li>Alfieri, K. A., Forsberg, J. A., Potter, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blast+injuries+and+heterotopic+ossification.">Blast injuries and heterotopic ossification.</a> Bone and Joint Research. 1 (8), 174-179 (2012).</li><li>Hunt, J. L., Arnoldo, B. D., Kowalske, K., Helm, P., Purdue, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+ossification+revisited:+a+21-year+surgical+experience.">Heterotopic ossification revisited: a 21-year surgical experience.</a> J Burn Care Res. 27 (4), 535-540 (2006).</li><li>Ring, D., Jupiter, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operative+release+of+ankylosis+of+the+elbow+due+to+heterotopic+ossification.+Surgical+technique.">Operative release of ankylosis of the elbow due to heterotopic ossification. Surgical technique.</a> J Bone Joint Surg Am. 86-A, 2-10 (2004).</li><li>Crane, N. J., Polfer, E., Elster, E. A., Potter, B. K., Forsberg, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raman+spectroscopic+analysis+of+combat-related+heterotopic+ossification.">Raman spectroscopic analysis of combat-related heterotopic ossification.</a> Bone. 57 (2), 335-342 (2013).</li></ol>

The authors have nothing to disclose

Heterotopic ossification represents a major functional impairment faced by patients that sustain trauma, burns, and invasive musculoskeletal procedures. The most at-risk population are soldiers in modern conflicts with major blast injuries from mechanisms such as improvised explosive devices (IED).18 Improved body armor and forward positioned medical units allows for improved survival of major extremity injury. After initial stabilization and repair of their extremity injury, these patients are at high risk...

Heterotopic ossification (HO) is the formation of ectopic bone in which osteo-potent cells are aberrantly induced to form endochondral bone outside of the skeleton. While the details of the HO formation pathway are still largely unknown, an accepted paradigm includes three key factors: an inflammatory inciting incident, a permissive niche, and mesenchymal stem cells capable of forming bone.1-3 HO is a common comorbidity complicating over 60% of major burn injuries, 65% of combat-related injuries, and 10% of invasive orthopaedic surgery cases.4,5 However, it is often difficult to predict where HO will form because it can occur at sites of local injury or at distant locations that may be otherwise uninjured. This variability in location makes it difficult to intervene prophylactically to prevent reactive bone formation in a locally targeted manner. There are also congenital forms of HO such as fibrodysplasia ossificans progressiva (FOP) in which patients are prone to the development of robust HO in response to minor trauma or inflammatory insult. Powerful animal models using transgenic mice have reproduced this phenotype and provided insight to the molecular pathways that may also be important in trauma induced HO.6-9 Translational research into the pathogenesis of non-congenital HO has used a wide variety of constructs ranging from injury alone to the implantation of exogenous osteo-inductive materials and/or cells.10-13
In our prior work we have validated a simple and reliable model of HO formation in mice which does not require the administration of any exogenous material.14-17 This model created two key conditions to initiate HO: local trauma and global inflammation. This was achieved through the use of an Achilles tenotomy (local trauma) combined with a distant burn injury (global inflammation). Mice received both treatments concurrently and were found to develop a robust amount of HO that could be analyzed by histologic, radiologic, and molecular means. Interestingly, concurrent burn injury significantly increased the amount of HO that formed and accelerated its developmental time-course.14-16 HO developed at predictable sites around the calcaneus, ankle joint, and tibia/fibula of the limb that received the tenotomy. The reliability of HO development at a known location allowed for focused examination of molecular and histologic features in the early stages of ectopic ossification.14,17 To date, 100% of mice (over 50 animals) with a tenotomy and concurrent burn injury have developed HO. Additionally, longitudinal 2D and 3D imaging and spectroscopic analysis were conducted to examine the growth pattern and biochemical make-up of HO.15,16

<table><tbody><tr><td>C57BL/6 mice</td><td>Jackson Laboratory</td><td>664</td><td>8-10 weeks old</td></tr><tr><td>Isoflurane &amp;ndash; Fluriso</td><td>VET one, Boise, ID</td><td>V1 501017</td><td></td></tr><tr><td>Buprenorphine &amp;ndash; Buprenex</td><td>Reckitt Benckiser Healthcare</td><td>NDC 12496-0757-1</td><td>0.3 mg/ml solution</td></tr><tr><td>Betadine</td><td>Owens and Minor, Mechanicsville, VA</td><td>2047PVP202</td><td></td></tr><tr><td>5-0 Vicryl sutures</td><td>Ethicon, Summerville, NJ</td><td>J493</td><td></td></tr><tr><td>Tegaderm Film, 6 cm x 7 cm</td><td>3M</td><td>1624W</td><td>Cut in half to properly cover burn site</td></tr><tr><td>&amp;micro;CT - GE eXplore Locus SP</td><td>GE Healthcare Pre-Clinical Imaging, London, ON, Canada</td><td></td><td></td></tr><tr><td>Microview 2.2 Advanced Bone Analysis Application</td><td>GE Healthcare Pre-Clinical Imaging, London, ON, Canada</td><td></td><td></td></tr></tbody></table>

direct mouse trauma/burn model of heterotopic ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

במחקר הנוכחי, את הפרוטוקול מתואר למודל העכבר שפורסם בעבר של טראומה / לשרוף מושרה HO. 14-17 זה כרוך ביצירה של פציעת שריר-שלד מקומי במקביל לtenotomy אכילס ועלבון דלקתי העולמי בעובי חלקי לשרוף פציעה. התוצאה היא היווצרות אמינה של עצם תגובה באתר tenotomy שניתן אחריו עם הדמיה סיד...

Watch this Scientific Journal Video about Direct Mouse Trauma/Burn Model of Heterotopic Ossification at JoVE.com

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

ישירה עכבר טראומה / צרוב דגם של Heterotopic התאבנות

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

direct-mouse-traumaburn-model-of-heterotopic-ossification

Research

JoVE Journal

Medicine

10.0K Views.  University of Michigan Medical School. An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

Video: Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Pseudofracture: An Acute Peripheral Tissue Trauma Model

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; this response is often accompanied by a delayed immunosuppression that adds the clinical complications of infection and can also increase mortality.1-9 Many studies have begun to assess these changes in the reactivity of the immune system following trauma.10-15 
Immunologic studies are greatly supported through the wide variety of transgenic and knockout mice available for in vivo modeling; these strains aid in detailed investigations to assess the molecular pathways involved in the immunologic responses.16-21
The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible.22-30
This pseudofracture model, an easily reproduced trauma model, overcomes these difficulties by immunologically mimicking an extremity fracture environment, while allowing freedom of movement in the animals and long term survival without the continual, prolonged use of anaesthesia. The intent is to recreate the features of long bone fracture; injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone.
The pseudofracture model consists of two parts: a bilateral muscle crush injury to the hindlimbs, followed by injection of a bone solution into these injured muscles. The bone solution is prepared by harvesting the long bones from both hindlimbs of an age- and weight-matched syngeneic donor. These bones are then crushed and resuspended in phosphate buffered saline to create the bone solution.
Bilateral femur fracture is a commonly used and well-established model of extremity trauma, and was the comparative model during the development of the pseudofracture model. Among the variety of available fracture models, we chose to use a closed method of fracture with soft tissue injury as our comparison to the pseudofracture, as we wanted a sterile yet proportionally severe peripheral tissue trauma model. 31
Hemorrhagic shock is a common finding in the setting of severe trauma, and the global hypoperfusion adds a very relevant element to a trauma model. 32-36 The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity. 37

Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma.

Pseudofracture: טראומה חריפה היקפיים דגם רקמות

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; ...

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

הזרקת תאי אוסטאוסרקומה אינטראטיביאלית להפקת אוסטאוסרקומה אורתוטופית ומודלים של עכבר גרורות ריאות

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

Cancer Research

A Mouse Distraction Osteogenesis Model

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the following three phases: the latency phase after osteotomy and placement of the external distractor; the distraction phase, wherein the separated bone ends are gradually and continuously distracted; and the consolidation phase. This custom-made distractor used for DO is comprised of two incomplete acrylic resin rings and an expansion screw. The process was initiated by making a mold with silicone impression material and then creating the custom-made distractor. Dental resin was poured into the formwork made of silicone impression material, and it was allowed to polymerize to create the incomplete resin rings required for the custom-made distractor. These rings were fixed with an expansion screw using transparent resin. The custom-made distractor created via this approach was attached to the tibia of mice. The tibia was fixed to the device using one pair of 25 G needles proximally, one pair of 27 G needles distally, and acrylic resin. After a latency period of 5 days, distraction was initiated at a rate of 0.2 mm/12 h. The lengthening was continued for 8 days, resulting in a total gap of 3.2 mm. The mice were sacrificed 4 weeks after distraction. Bone formation in the distraction gap was confirmed using both radiography and histology.

We present a mouse tibial distraction osteogenesis model developed using a custom-made distractor. The use of a mouse as an analysis target is advantageous for advancing research.

מודל פרכת העכבר הסחת דעת

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the ...

Developmental Biology

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.

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.

כריתה של ספרות עכבר למבוגרים והתחדשות: מודל פשוט כדי לחקור ממגי בלסטאמה היווצרות הIntramembranous פיקציה

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

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.

דגם פגם עצם הירך קריטית בגודל פשוט בעכברים

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 Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

דגם העכבר של כירורגיה אורתופדית ללמוד לאחר הניתוח בתפקוד הקוגניטיבי ו התחדשות רקמות

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

A Mouse Model of Mechanotransduction-driven, Human-like Hypertrophic Scarring

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have demonstrated that mechanotransduction&#8212;the conversion of mechanical stimuli into cellular responses&#8212;drives excessive fibrotic scar healing. A mouse model to assess human-like hypertrophic scarring would be an essential tool for examining various therapeutics and their ability to reduce scarring and improve healing. Specifically, our laboratory has developed a murine wound model that increases mechanical strain to promote human-like HTS. This&#160;protocol utilizes biomechanical loading devices, made from modified 13 mm palatal expanders, whose arms are placed on either side of the incision and distracted incrementally apart in order to apply continuous tension across the wound bed during healing. Over nearly two decades of use, this model has been significantly advanced to improve efficacy and reproducibility. Using the murine HTS model, significant dermal fibrotic scars can be induced to be histologically comparable to human hypertrophic scars. This murine model provides an environment to develop biologics involved in the treatment of HTS and mechanotransduction-related conditions such as foreign body response.

This protocol will explain how to establish a hypertrophic scarring murine model that increases mechanotransduction signaling to simulate human-like scarring. This method involves increasing mechanical tension across a healing incision in a mouse and using a specialized device to create reproducible, excessive scar tissue for detailed histological and bioinformatic analyses.

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have ...

Bioengineering

The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue damage that manifests with this type of injury in humans. This model was developed because previous models of orthopedic trauma did not include simultaneous injury to multiple tissue types (bone, muscle, nerves) and were not truly representative of human complex orthopedic trauma. The authors therefore modified previous models of orthopedic trauma and developed the tibial fracture-pin model. This modified fracture model consists of a unilateral open tibial fracture with intramedullary nail (IMN) internal fixation and simultaneous tibialis anterior (TA) muscle injury, resulting in mechanical allodynia that lasts up to 5 weeks post injury. This series of protocols outlines the detailed steps to perform the clinically relevant orthopedic trauma tibial fracture-pin model, followed by a modified hot plate assay to examine nociceptive changes after orthopedic injury. Taken together, these detailed, reproducible protocols will allow pain researchers to expand their toolkit for studying orthopedic trauma-induced pain.

The tibial fracture-pin model is a clinically relevant model of orthopedic trauma comprising a unilateral open tibial fracture with intramedullary nail internal fixation and simultaneous injury to the tibialis anterior muscle. Thermal sensitivity in this model can be measured using a 45 s hot plate paradigm.

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue ...

Neuroscience

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

מודל מורנה של פצע צריבה שוחזר עם שתל העור Allogeneic

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

Biology

ניאו-וסקולריזציה של הקרנית הנגרמת על ידי כוויות אלקליות בעכברים

A Mouse Model for Corneal Neovascularization by Alkali Burn

Corneal neovascularization (CoNV), a pathological form of angiogenesis, involves the growth of blood and lymph vessels into the avascular cornea from the limbus&#160;and adversely&#160;affects transparency and vision. Alkali burn is one of the most common forms of ocular trauma that leads to CoNV. In this protocol, CoNV is experimentally induced using sodium hydroxide solution in a controlled manner to ensure reproducibility. The alkali burn model is useful for understanding the pathology of CoNV and can be extended to study angiogenesis in general because of the avascularity, transparency, and accessibility of the cornea. In this work, CoNV was analyzed by direct examination under a dissecting microscope and by immunostaining flat-mount corneas using anti-CD31 mAb. Lymphangiogenesis was detected on flat-mount corneas by immunostaining using anti-LYVE-1 mAb. Corneal edema was visualized and quantified using optical coherence tomography (OCT). In summary, this model will help to advance existing neovascularization assays and discover new treatment strategies for pathologic ocular and extraocular angiogenesis.

מחבר זרקור: פענוח ניאו-וסקולריזציה של הקרנית עם מודל כוויות אלקליות לאסטרטגיות טיפוליות עתידיות 

This protocol focuses on alkali burn-induced corneal neovascularization in mice. The method generates a reproducible and controllable corneal disease model to study pathological angiogenesis and the associated molecular mechanisms and to test new pharmacological agents to prevent corneal neovascularization.

מודל עכבר לניאו-וסקולריזציה של הקרנית על ידי כוויה אלקלית

Corneal neovascularization (CoNV), a pathological form of angiogenesis, involves the growth of blood and lymph vessels into the avascular cornea from the ...