The authors would like to thank Central Council for Research in Homoeopathy (CCRH), Ministry of AYUSH, Govt. of India, for research funding. The authors are grateful for the help and support of Central Animal Facility, AIIMS, New Delhi, for their help and support with the animal experiments and CMET, AIIMS, New Delhi, for their help and support in photography and videography.

Animal experiments were done after taking ethical approval from the Institutional Animal Ethics Committee (IAEC), AIIMS, New Delhi, India (286/IAEC-1/2021).
1. Preoperative procedure
<ol>
	<li>House male Wistar albino rats 6-8 weeks of age, weighing between 150-200 g each, at a Central Animal Facility (CAF) in separate individual cages. This ensures no surgical/fracture site injury when multiple rats share cages.</li>
	<li>Keep the rats at a temperature of 23 &#176;C &#177; 2 &#176;C in a humidity-controlled environment with a relative humidity of 50% &#177; 5%, expose them to a 12 h dark/light cycle, and give ad libitum access to food (standard semi-synthetic diet): pellet diet (dry) and water. The composition of the standard semi-synthetic diet is as follows: roasted Bengal gram flour (60%), wheat flour (22%), casein (4%), skim milk powder (5%), refined oil (4%), salt mixture with starch (4.8%), and vitamin choline mixture with starch (0.2%).</li>
	<li>Acclimatize the rats for a period of at least 48 h before surgery.</li>
	<li>Weigh each rat on a digital weighing scale and note the weight.</li>
	<li>Administer intraperitoneal (IP) injections of cefuroxime (100 mg/kg body weight), tramadol (25 mg/kg body weight), and a combination of ketamine (75 mg/kg body weight) with xylazine (10 mg/kg body weight) to the rats 15 min before starting the surgical procedure. Apply ophthalmic ointment to both eyes to prevent dry eye.</li>
	<li>Remove the hair from the right lower limb, from the flank region up to the knee joint, with topical application of a hair removal cream. 
	&#8203;NOTE: Blood (0.5 mL) can be collected from the tail vein of each rat for the baseline analysis of different parameters. Blood can be collected again every 2 weeks after the surgery.</li>
</ol>
2. Surgical procedure for creating complete transverse fracture through open osteotomy
NOTE: Use a designated operation room with an operating table and optimal ambient temperature (26 &#176;C) for performing the procedure.
<ol>
	<li>Place the wax block (aluminum tray 30 cm x 30 cm x 4 cm containing wax up to a depth of 2.5 cm) on the operating table and cover it with sterile drapes. The wax block prevents any change in the animal&#39;s position during surgery.</li>
	<li>Confirm the onset of anesthesia (by checking loss of toe pinch). Place the anesthetized rat on a sterile drape in the left lateral position. Ask an assistant to hold the right lower limb (knee and hip) in extension. Keep a sterile hard support (marble block) under the right leg to support the femur. Clean the surgical site with alcohol and betadine.</li>
	<li>Inject local anesthesia (0.25 mL of 1% lignocaine) at the site of incision (lateral aspect of the right thigh), cut a hole in another sterile drape, and expose only the right leg of the rat through it for surgery.</li>
	<li>Give a 1 cm vertical skin incision on the lateral side of the right thigh, and extend it as per need with a no. 15 surgical blade.</li>
	<li>Expose the vastus lateralis muscle by separating the deep fascia using Metzenbaum scissors. Split the vastus lateralis in line with the muscle fibers using artery forceps until the shaft of the femur is reached.</li>
	<li>Free the bone from the muscles attached to it using the periosteal elevator.</li>
	<li>Inject local anesthesia (0.2 mL of 1% lignocaine) in and around the periosteum to prevent vasovagal reflex.</li>
	<li>Create an indentation in the middle third of the shaft of the femur using the no.15 surgical blade, and fracture the bone in the middle-third of the shaft (complete fracture) by placing a chisel on the indentation made (so that the chisel does not slip) and gently tapping the chisel with a hammer. Use the sterile hard support (marble block) to support the bone while fracturing it to ensure a clean break. 
	NOTE: The sterile hard support usually does not cause a significant injury to the muscles underneath.</li>
	<li>Internally fix the fracture using a sterile K-wire (1.0 mm) held with the help of a battery-operated power drill. Pass the K-wire into the medullary canal of the distal fragment through the fracture site. Then, drill out the K-wire through the distal end of the femur using the battery-operated power drill. 
	NOTE: Disinfect the surface of the power drill with alcohol before use. Change gloves after the K-wire is fixed.</li>
	<li>After reducing the fracture, advance the K-wire from the distal end into the canal of the proximal fragment until it obtains purchase in the trochanteric region. Cut off the distal part of the K-wire protruding through the skin using a wire cutter.</li>
	<li>Bend the tip of the K-wire to around 90&#176; using pliers and use a gauze bandage soaked in betadine for pin-site dressing. The K-wire acts as an intramedullary splint for keeping the fracture in a reduced position.</li>
	<li>Ensure complete hemostasis before closing the skin using a 3-0 nylon suture. Apply pressure on the bleeding area using sterile gauze or artery forceps to stop any bleeding.</li>
	<li>Clean the wound with betadine, and cover it with sterile gauze and micropore adhesive tape.</li>
</ol>
3. Postoperative care
<ol>
	<li>Return the rats to their cages, allow normal ambulation, and continue giving a standard semi-synthetic diet until sacrificing them, as well as antibiotics (injection cefuroxime 100 mg/kg) and analgesics (injection tramadol 25 mg/kg/day in two divided doses) intraperitoneally for 5 days after the procedure. 
	NOTE: The rats can be divided into treatment and control groups to test a particular drug. If the drug is water soluble, it can be given orally through gavage. The weight of the individual animals may be noted to calculate the dose of the drug to be used. Inclusion and exclusion criteria can be followed to ensure homogeneity of the animal groups.</li>
	<li>House the animals in individual cages under similar conditions to the preoperative period. Inspect the surgical site every day to look for any signs of postoperative pain, wound infection, slipping of sutures, or any abdominal swelling or discomfort.</li>
	<li>Assess bone healing by X-ray of the fractured site once weekly.</li>
</ol>
4. Radiological procedure
<ol>
	<li>Before the X-ray, anesthetize the rats with an intraperitoneal injection of ketamine (50 mg/kg body weight) and xylazine (5 mg/kg body weight).</li>
	<li>Keep the hip joint of the rat in a flexed and abducted position while the knee joint is kept semi-flexed to take the X-ray of the fractured limb with the following exposure settings: Ref. kVp &#8776; 62; Ref. mAS = 6.4; and automatic exposure settings (Ref. mA=160). 
	&#8203;NOTE: X-rays were taken at baseline (1 day after surgery) and then once weekly until sacrifice or 5 weeks.</li>
</ol>
5. Animal euthanasia and callus retrieval
<ol>
	<li>Sacrifice the rats by an overdose of carbon dioxide (administer 100% CO2 at a flow rate of 7-8 L/min for 1 min, followed by a waiting period of 4-5 min), at two previously determined time points, based on the radiological appearance of soft and hard bridging calluses, respectively.</li>
	<li>Incise the skin parallel to the femur and separate the overlying muscles carefully to avoid damage to the callus tissue.</li>
	<li>Fracture the bone between the hip joint and callus tissue using a hammer and chisel. Similarly, fracture the bone between the callus and the knee joint. Remove the K-wire and clean the bone piece in saline to remove blood clots and soft tissue.</li>
	<li>Transfer the callus immediately to a labeled container with 10% neutral buffered formalin (20 mL per sample) and keep it for 3 days at room temperature (RT).</li>
</ol>
6. Decalcification of bone and callus tissue
<ol>
	<li>Take the callus tissue from formalin and keep it at RT in 20% ETDA solution, pH 7, for decalcification of the bone tissue.</li>
	<li>Change fresh EDTA solution every 2 days for approximately 3 weeks, and check for bone decalcification by poking the bone with a needle without disturbing the callus tissue. Optimal decalcification is denoted by the loss of the normal gritty sensation of the bone tissue.</li>
	<li>After complete decalcification, cut the sagittal section of the callus and prepare paraffin blocks of the callus tissue. Cut 4 &#181;m thick sections of the callus tissue for histopathological16 and any other comparative analysis17.</li>
</ol>

<ol>
	<li>Wang, T., Zhang, X., Bikle, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+differentiation+of+periosteal+cells+during+fracture+healing.">Osteogenic differentiation of periosteal cells during fracture healing.</a> Journal of Cellular Physiology. 232 (5), 913-921 (2017).</li><li>Fakhry, M., Hamade, E., Badran, B., Buchet, R., Magne, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+mesenchymal+stem+cell+differentiation+towards+osteoblasts.">Molecular mechanisms of mesenchymal stem cell differentiation towards osteoblasts.</a> World Journal of Stem Cells. 5 (4), 136-148 (2013).</li><li>Bishop, J. A., Palanca, A. A., Bellino, M. J., Lowenberg, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+compromised+fracture+healing.">Assessment of compromised fracture healing.</a> JAAOS - Journal of the American Academy of Orthopaedic Surgeons. 20 (5), 273-282 (2012).</li><li>Fong, 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=Predictors+of+nonunion+and+reoperation+in+patients+with+fractures+of+the+tibia:+an+observational+study.">Predictors of nonunion and reoperation in patients with fractures of the tibia: an observational study.</a> BMC Musculoskeletal Disorders. 14 (1), 103 (2013).</li><li>Ramoutar, D. N., Rodrigues, J., Quah, C., Boulton, C., Moran, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Judet+decortication+and+compression+plate+fixation+of+long+bone+nonunion:+Is+bone+graft+necessary.">Judet decortication and compression plate fixation of long bone nonunion: Is bone graft necessary.</a> Injury. 42 (12), 1430-1434 (2011).</li><li>Goulet, J. A., Senunas, L. E., DeSilva, G. L., Greenfield, M. L. V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autogenous+iliac+crest+bone+graft:+Complications+and+functional+assessment.">Autogenous iliac crest bone graft: Complications and functional assessment.</a> Clinical Orthopaedics and Related Research. 339, 76-81 (1997).</li><li>Stevenson, 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=A+systematic+review+and+economic+evaluation+of+alendronate,+etidronate,+risedronate,+raloxifene+and+teriparatide+for+the+prevention+and+treatment+of+postmenopausal+osteoporosis.">A systematic review and economic evaluation of alendronate, etidronate, risedronate, raloxifene and teriparatide for the prevention and treatment of postmenopausal osteoporosis.</a> Health Technology Assessment. 9 (22), 1 (2005).</li><li>Haffner-Luntzer, M., Kovtun, A., Rapp, A. E., Ignatius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+in+bone+fracture+healing+research.">Mouse models in bone fracture healing research.</a> Current Molecular Biology Reports. 2 (2), 101-111 (2016).</li><li>Mills, L. A., Simpson, A. H. R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+models+of+bone+repair.">In vivo models of bone repair.</a> The Journal of Bone and Joint Surgery. British Volume. 94 (7), 865-874 (2012).</li><li>Houdebine, L. -. M., Sioud, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+Animal+Models+in+Biomedical+Research.">Transgenic Animal Models in Biomedical Research.</a> Target Discovery and Validation Reviews and Protocols: Volume 1, Emerging Strategies for Targets and Biomarker Discovery. , (2007).</li><li>Histing, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+bone+healing+models:+Standards,+tips+and+pitfalls+results+of+a+consensus+meeting.">Small animal bone healing models: Standards, tips and pitfalls results of a consensus meeting.</a> Bone. 49 (4), 591-599 (2011).</li><li>Bonnarens, F., Einhorn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+standard+closed+fracture+in+laboratory+animal+bone.">Production of a standard closed fracture in laboratory animal bone.</a> Journal of Orthopaedic Research. 2 (1), 97-101 (1984).</li><li>Klein, 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=Comparison+of+healing+process+in+open+osteotomy+model+and+open+fracture+model:+delayed+healing+of+osteotomies+after+intramedullary+screw+fixation.">Comparison of healing process in open osteotomy model and open fracture model: delayed healing of osteotomies after intramedullary screw fixation.</a> Journal of Orthopaedic Research. 33 (7), 971-978 (2015).</li><li>Kolios, 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=Do+estrogen+and+alendronate+improve+metaphyseal+fracture+healing+when+applied+as+osteoporosis+prophylaxis.">Do estrogen and alendronate improve metaphyseal fracture healing when applied as osteoporosis prophylaxis.</a> Calcified Tissue International. 86 (1), 23-32 (2010).</li><li>Holstein, J. H., 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=Advances+in+the+establishment+of+defined+mouse+models+for+the+study+of+fracture+healing+and+bone+regeneration.">Advances in the establishment of defined mouse models for the study of fracture healing and bone regeneration.</a> Journal of Orthopaedic Trauma. 23, 31-38 (2009).</li><li>Umiatin, U., Dilogo, I. H., Sari, P., Wijaya, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+analysis+of+bone+callus+in+delayed+union+model+fracture+healing+stimulated+with+pulsed+electromagnetic+fields+(PEMF).">Histological analysis of bone callus in delayed union model fracture healing stimulated with pulsed electromagnetic fields (PEMF).</a> Scientifica. 2021, 4791172 (2021).</li><li>Han, W., 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=The+osteogenic+potential+of+human+bone+callus.">The osteogenic potential of human bone callus.</a> Scientific Reports. 6, 36330 (2016).</li><li>Haffner-Luntzer, 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=A+novel+mouse+model+to+study+fracture+healing+of+the+proximal+femur.">A novel mouse model to study fracture healing of the proximal femur.</a> Journal of Orthopaedic Research. 38 (10), 2131-2138 (2020).</li><li>Aur&#233;gan, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+model+of+femur+fracture+for+bone+and+mineral+research:+An+improved+description+of+expected+comminution,+quantity+of+soft+callus+and+incidence+of+complications.">The rat model of femur fracture for bone and mineral research: An improved description of expected comminution, quantity of soft callus and incidence of complications.</a> Bone &amp; Joint Research. 2 (8), 149-154 (2013).</li><li>Li, Z., Helms, 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=Drill+hole+models+to+investigate+bone+repair.">Drill hole models to investigate bone repair.</a> Methods in Molecular Biology. 2221, 193-204 (2021).</li><li>Handool, K. 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=Optimization+of+a+closed+rat+tibial+fracture+model.">Optimization of a closed rat tibial fracture model.</a> Journal of Experimental Orthopaedics. 5 (1), 13 (2018).</li><li>Kobata, S. I., 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=Prevention+of+bone+infection+after+open+fracture+using+a+chitosan+with+ciprofloxacin+implant+in+animal+model.">Prevention of bone infection after open fracture using a chitosan with ciprofloxacin implant in animal model.</a> Acta Cirurgica Brasileira. 35 (8), 202000803 (2020).</li></ol>

None of the authors have any conflicts of interest or any other financial disclosures.

This method lucidly describes the details needed to develop a fracture osteotomy model in Wistar albino rats. This model can be used to evaluate the osteogenic effect of a promising osteoanabolic drug in fracture healing, as well as understand the intricacies of bone healing. The salient feature of this method is that it is simple and does not need too much time or sophisticated equipment. In this method, adult male Wistar albino rats were selected as the rodent model for the experiments. Uniform gender was selected to r...

Bone is a dense connective tissue consisting of bone-forming cells, the osteoblasts, and bone-resorbing cells, the osteoclasts. Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts1. When there is a fracture, osteoblastic and osteoclastic activity at the fracture site are some of the important factors that determine bone healing2. When fracture healing deviates from its normal course, it results in a delayed union, malunion, or nonunion. A fracture is said to be in nonunion when there is a failure of union of the fracture for 9 months, with no progression of repair in the last 3 months3. Approximately 10%-15% of all fractures experience a delay in repair that may progress to nonunion4. The nonunion rate for all fractures is 5%-10% and varies depending on the bone involved and the site of fracture5.
The current regimen for the treatment of fracture nonunion comprises surgical and/or medical modalities. Currently, delayed or nonunion of fractures can be overcome by surgical strategies like bone grafting. However, bone grafting has its limitations and complications like availability of graft tissue, donor site pain, morbidity, and infection6. Medical treatment comprises osteoanabolic drugs like bone morphogenetic protein (BMP) and teriparatide (parathormone analog). Currently used osteoanabolic agents have the potential to augment the repair of fractures but have constraints like exorbitant costs or undesirable side effects7. Hence, there is scope for identifying cost-effective, nonsurgical alternatives for bone healing. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. A drug that is known to enhance bone healing should be evaluated in vitro and, if found promising, can be used for in vivo animal model studies. If the drug proves to promote bone formation and remodeling in the in vivo model, it could proceed to the next stage (i.e., clinical trials).
Assessing fracture healing in animals is a logical step forward to evaluate a novel agent introduced for bone healing before it undergoes human trials. For in vivo animal model studies of fracture healing, rodents have become an increasingly popular model8. The rodent models have generated increasing interest due to the low operational costs, limited need for space, and less time needed for bone healing9. In addition, rodents have a broad spectrum of antibodies and gene targets, which allow studies on the molecular mechanisms of bone healing and regeneration10. A consensus meeting comprehensively highlighted various small animal bone healing models and focused on the different parameters influencing bone healing, as well as emphasizing several small animal fracture models and implants11.
Basic fracture models can be broadly divided into open or closed models. Closed fracture models use a three- or four-point bending force on the bone and do not require a conventional surgical approach. They lead to oblique or spiral fractures, resembling long bone fractures in humans, but the lack of standardization of fracture location and dimensions may act as a confounding factor in them12. Open fracture models require surgical access for osteotomy of the bone, help to achieve a more consistent fracture pattern at the fracture site, but are associated with delayed healing compared to the closed models13. The choice of bone used to study fracture healing mainly remains the tibia and femur due to their dimensions and accessibility. The choice of the site of fracture is usually the diaphysis or metaphysis. The metaphyseal region is specially chosen in cases where fracture healing is studied in osteoporotic subjects, as the metaphysis is more affected by osteoporosis14. Several implants like intramedullary pins and external fixators can be used to stabilize the fracture11,15.
The objective of this study was to develop a simple and easy-to-follow rodent model that could help researchers not only understand the development of the callus following fracture of the femur but could help to determine whether a potential drug has bone healing properties by understanding the mechanism by which it acts.

<table><tbody><tr><td>Alcohol</td><td>Raman &amp;amp; Weil Pvt. Ltd, Mumbai, Maharashtra, India</td><td>MFG/MD/2019/000189</td><td>Sterillium hand disinfectant</td></tr><tr><td>Artery forceps&amp;nbsp;</td><td>Nebula surgical, Gujarat, India</td><td>G.105.05S</td><td>5&quot;, straight</td></tr><tr><td>Bard-Parker handle&amp;nbsp;</td><td>Nebula surgical, Gujarat, India</td><td>G.103.03</td><td>Size number 3</td></tr><tr><td>Betadine solution</td><td>Win-medicare New Delhi, India</td><td>UP14250000001</td><td>10% w/v Povidone iodine solution</td></tr><tr><td>Cat&#039;s-paw skin retractor&amp;nbsp;</td><td>Nebula surgical, Gujarat, India</td><td>908.S</td><td>Small</td></tr><tr><td>EDTA</td><td>Sisco research laboratories Pvt. Ltd, Maharashtra, India</td><td>43272</td><td>Disodium salt</td></tr><tr><td>Eosin</td><td>Sigma Aldrich, Merck Life Sciences Pvt Ltd, Mumbai, Maharashtra, India</td><td>115935</td><td>For preparing the staining solution&amp;nbsp;</td></tr><tr><td>Forceps (plain)</td><td>Nebula surgical, Gujarat, India</td><td>115.06</td><td>6&quot;, plain</td></tr><tr><td>Forceps (toothed)</td><td>Nebula surgical, Gujarat, India</td><td>117.06</td><td>6&quot;, toothed</td></tr><tr><td>Formaldehyde</td><td>Sisco research laboratories Pvt. Ltd, Maharashtra, India</td><td>84439</td><td>For preparing the neutral buffered formalin&amp;nbsp;</td></tr><tr><td>Haematoxylin</td><td>Sigma Aldrich, Merck Life Sciences Pvt Ltd, Mumbai, Maharashtra, India</td><td>104302</td><td>For preparing the staining solution&amp;nbsp;</td></tr><tr><td>Hammer</td><td>Nebula surgical, Gujarat, India</td><td>401.M</td><td></td></tr><tr><td>Injection Cefuroxime</td><td>Akumentis Healthcare Ltd, Thane, Maharashtra, India</td><td>48/UA/SC/P-2013</td><td>Cefuroxime sodium IP, 1.5 g/vial&amp;nbsp;</td></tr><tr><td>Injection Ketamine</td><td>Baxter Pharmaceuticals India Private Limited, Gujarat, India</td><td>G/28-B/6</td><td>Ketamine hydrochloride IP, 50 mg/mL&amp;nbsp;</td></tr><tr><td>Injection Xylazine</td><td>Indian Immunologicals Limited, Hyderabad, Telangana, India</td><td>28/RR/AP/2009/F/G</td><td>Xylazine hydrochloride USP, 20 mg/mL</td></tr><tr><td>Injection Lignocaine</td><td>Jackson laboratories Pvt Limited, Punjab, India&amp;nbsp;</td><td>1308-B</td><td>2% Lignocaine Hydrochloride IP, 21.3 mg/mL</td></tr><tr><td>Injection Tramadol&amp;nbsp;</td><td>Intas Pharmaceuticals Limited, Ahmedabad, Gujarat, India</td><td>MB/07/500</td><td>Tramadol hydrochloride IP, 50 mg/mL</td></tr><tr><td>K-wire&amp;nbsp;</td><td>Nebula surgical, Gujarat, India</td><td>166 (1mm)</td><td>12&quot;, double ended</td></tr><tr><td>Mechanical drill for inserting K-wire</td><td>&amp;lrm;Bosch, Germany&amp;nbsp;</td><td>06019F70K4</td><td>GSR 120-LI Professional</td></tr><tr><td>Metzenbaum cutting scissors&amp;nbsp;</td><td>Nebula surgical, Gujarat, India</td><td>G.121.06S</td><td>6&quot;, straight</td></tr><tr><td>Needle holder</td><td>Nebula surgical, Gujarat, India</td><td>G.108.06</td><td>6&quot;, straight</td></tr><tr><td>Ophthalmic ointment&amp;nbsp;</td><td>GlaxoSmithKline Pharmaceutical Limited, Bengaluru, Karnataka, India</td><td>KTK/28a/467/2001</td><td>Neomycin, Polymixin B sulfate and Bacitracin zinc ophthalmic ointment USP</td></tr><tr><td>Osteotome (chisel)</td><td>Nebula surgical, Gujarat, India</td><td>1001.S.10</td><td>10 mm, straight</td></tr><tr><td>Periosteal elevator&amp;nbsp;</td><td>Nebula surgical, Gujarat, India</td><td>918.10.S</td><td>10 mm, straight</td></tr><tr><td>Pliers cum wire cutter</td><td>Nebula surgical, Gujarat, India</td><td>604.65</td><td></td></tr><tr><td>Reynold&amp;rsquo;s scissors</td><td>Nebula surgical, Gujarat, India</td><td>G.110.06S</td><td>6&quot;, straight</td></tr><tr><td>Standard semi-synthetic diet&amp;nbsp;</td><td>Ashirvad Industries, Chandigarh, India</td><td>No catalog number available</td><td>Detailed composition provided in materials used</td></tr><tr><td>Steel cup for keeping betadine for application</td><td>Local purchase</td><td>No catalog number available</td><td></td></tr><tr><td>Steel tray with lid for autoclaving instruments</td><td>Local purchase</td><td>No catalog number available</td><td></td></tr><tr><td>Sterile gauze</td><td>Ideal Healthcare Industries, Delhi, India&amp;nbsp;</td><td>E(0047)/14/MNB/7951</td><td>Sterile, 5cmx5cm, 12 ply</td></tr><tr><td>Sterile marble block for support</td><td>Local purchase</td><td>No catalog number available</td><td>Locally fabricated; autoclavable</td></tr><tr><td>Syringe and needle (1 mL)&amp;nbsp;</td><td>Becton Dickinson India Pvt. Ltd., Haryana, India</td><td>REF 303060</td><td>1 mL sterile Syringe with 26 G x 1/2 (0.45 mm x 13 mm) needle</td></tr><tr><td>Syringe and needle (2 mL)&amp;nbsp;</td><td>Becton Dickinson India Pvt. Ltd., Haryana, India</td><td>REF 307749</td><td>2 mL sterile syringe with 24 G x 1&#039;&#039; (0.55 mm x 25 mm) needle</td></tr><tr><td>Syringe and needle (10 mL)&amp;nbsp;</td><td>Hindustan Syringes &amp;amp; Medical Devices Ltd. Faridabad, India&amp;nbsp;</td><td>334-B(H)</td><td>10 mL sterile syringe with 21 G x1.5&quot; (0.80 mm x 38 mm) needle</td></tr><tr><td>Surgical blades (size no.15)</td><td>Paramount Surgimed Ltd, New Delhi, India for Medline Industries Inc, IL, USA</td><td>REF MDS15115E</td><td>Sterile, Single use</td></tr><tr><td>Surgical blades (size no.24)</td><td>Paramount Surgimed Ltd, New Delhi, India for Medline Industries Inc, IL, USA</td><td>REF MDS15124E</td><td>Sterile, Single use</td></tr><tr><td>Sutures</td><td>Healthium Medtech Pvt Ltd, Bangalore, Karnataka, India</td><td>SN 3318</td><td>4-0, 16 mm, 3/8 circle cutting needle, monofilament polyamide suture&amp;nbsp;</td></tr><tr><td>Wax block in aluminium tray&amp;nbsp;</td><td>Locally fabricated</td><td>No catalog number available</td><td>30 cm x 30 cm x 4 cm aluminium tray containing wax (to prevent animal from slipping)</td></tr><tr><td>X-ray machine</td><td>Philips India Ltd, Gurugram, Haryana</td><td>SN19861013</td><td>Model: Philips Digital Diagnost R 4.2&amp;nbsp;</td></tr></tbody></table>

protocol for developing a femur osteotomy model in wistar albino rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

This study was undertaken to develop a femur osteotomy model in Wistar albino rats. This model can be used to evaluate bone healing, as well as the osteogenic effect of a promising osteoanabolic drug in bone healing. Standard surgical precautions and protocols were followed. Sterile gowns, drapes, and surgical equipment were used for the procedure (Figure 1). The equipment (Table 1) was sterilized 48 h before surgery. Anesthetic, analgesic, and antibiotics were used as per t...

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Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

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Research

JoVE Journal

Medicine

2.7K Views.  All India Institute of Medical Sciences (AIIMS). Dit femur osteotomiemodel kan onderzoekers helpen het proces van fractuurgenezing te bestuderen en te evalueren hoe een medicijn de genezing van fracturen kan beïnvloeden. Het is een eenvoudig en gemakkelijk te volgen knaagdiermodel dat onderzoekers kan helpen identificeren of een medicijn botgenezende eigenschappen heeft en het werkingsmechanisme ervan te begrijpen. Hoewel het zich vooral richt op het observeren en analyseren van fractuurgenezing, kan het ook inzicht geven in de pathogenese...

Video: Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

One constraint of preclinical research in the field of bone repair is the lack of experimental control over the local mechanical environment within a healing bone lesion. We report the design and use of an external fixator for bone repair with the ability to change fixator stiffness in vivo.

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there ...

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (&#181;CT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent &#181;CT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ...

Bioengineering

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

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 ...

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired ...

Establishment and Evaluation of a Rat Femoral Box: Cavity Defect Model

Creating a Box-Cavity Defect Model in the Cortical Bone of Rat Femora

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which involves the application of cells, scaffolds, and cytokines, is considered a promising solution for bone regeneration. Consequently, various animal models that simulate bone defects play a crucial role in exploring the therapeutic potential of tissue engineering for bone healing. In this study, we established a box-shaped cortical bone defect model in the mid-femur of rats, which could serve as an ideal model for assessing the function of biomaterials in promoting bone healing. This box-shaped cortical bone defect was drilled using an oral low-speed handpiece and shaped by a lathe needle. Post-operative micro-CT analysis was immediately conducted to confirm the successful establishment of the box-cavity cortical bone defect. The femurs on the operated side of the rats were then harvested at multiple time points post-surgery (0 days, 2 weeks, 4 weeks, and 6 weeks). The healing process of each sample&#39;s defect area was evaluated using micro-CT, hematoxylin and eosin (H&#38;E) staining, and Masson trichrome staining. These results demonstrated a healing pattern consistent with intramembranous ossification, with healing essentially complete by 6 weeks. The categorization of this animal model&#39;s healing process provides an effective in vivo method for investigating novel biomaterials and drugs that target intramembranous ossification during bone tissue defect healing.

Author Spotlight: The Box-Cavity Cortical Approach for Enhanced Evaluation of Biomaterials and Bone Regeneration

Here, we present a protocol for creating a box-cavity defect in rat femoral diaphysis tissue. This model can assess biomaterial performance under biomechanical stress and explore mechanisms of bone regeneration related to intramembranous osteogenesis.

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which ...

Surgical Bone Implantation Technique for Rat Tibia Models of Diabetes and Osteoporosis

The rat has long served as a valuable animal model in implant dentistry and orthopedics, particularly in studying the interactions between biomaterials and bone tissue. The rat's tibia is frequently chosen due to its easy surgical access through thin tissue layers (skin and muscle) and the flattened shape of its medial face, facilitating the surgical insertion of intraosseous devices. Additionally, this model enables the induction of specific diseases, mimicking various clinical conditions to assess biological responses to different implant conditions like geometry, surface texture, or biological cues. However, despite its robust cortical structure, certain intraosseous devices may require adaptations in design and size for successful implantation. Therefore, establishing standardized surgical methods for manipulating both soft and hard tissues in the implantation region is essential for ensuring proper implant or screw device placement, particularly in fields like implant dentistry and orthopedics. This study included eighty Sprague Dawley rats divided into two groups based on their respective diseases: Group 1 with osteoporosis and Group 2 with Type 2 Diabetes. Implantations were performed at 4 weeks and 12 weeks, with the same surgeon following a consistent surgical technique. A positive biological response was observed, indicating complete osseointegration of all implants placed. These results validate the success of the surgical protocol, which can be replicated for other studies and serve as a benchmark for the biomaterials community. Notably, osseointegration values remained stable at both 4 weeks and 12 weeks for both disease models, demonstrating a durable integration of the implant over time and emphasizing the establishment of an intimate bone connection as early as 4 weeks.

The placement of implants in a rat model is an essential experimental procedure for clinical research. This study presents a comprehensive surgical protocol for implanting titanium implants into the tibia of rat models with diabetes and osteoporosis.

The rat has long served as a valuable animal model in implant dentistry and orthopedics, particularly in studying the interactions between biomaterials ...

Elastic Tape Rat Model of Steroid-Induced Femoral Head Osteonecrosis Enhancement

Tension-Free Weight-Bearing Model of Steroid-Induced Osteonecrosis of Femoral Head in Rats

Unlike humans, rats are animals that walk on all fours, while humans are bipedal animals that stand, and the hips are subjected to tremendous pressure when walking and standing. In rat steroid-induced femoral head necrosis models, the biomechanical characteristics of the human hip under higher pressure often need to be simulated. Some scholars try to emulate the state of human hip pressure by making rats bear a certain weight, but fixing the weight-bearing object on the rat is tough. Rats can easily break free of immobilization, and sticking the weight on the rats with adhesive tape will cause the rats to suffocate or die from intestinal obstruction. Our research group used elastic therapeutic tape to perform tension-free immobilization of weight-bearing objects in rats so that the rats could breathe freely and not break away from the immobilization under weight-bearing conditions. Compared to the usual steroid-induced femoral head necrosis rat model, we found that this weight-bearing intervention can aggravate the progression of femoral head necrosis in rats.

Author Spotlight: Developing a Rat Model for Weight-Bearing Intervention to Investigate Osteonecrosis of the Femoral Head

The protocol shows building a simple and cost-effective rat weight-bearing training model for steroid-induced osteonecrosis of the femoral head using elastic therapeutic tape.

Unlike humans, rats are animals that walk on all fours, while humans are bipedal animals that stand, and the hips are subjected to tremendous pressure ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

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