Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Summary

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.

Abstract

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.

Introduction

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 fracture^11,15.

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.

Protocol

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

1. 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.
2. Keep the rats at a temperature of 23 °C ± 2 °C in a humidity-controlled environment with a relative humidity of 50% ± 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%).
3. Acclimatize the rats for a period of at least 48 h before surgery.
4. Weigh each rat on a digital weighing scale and note the weight.
5. 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.
6. 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.
   ​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.

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 °C) for performing the procedure.

1. 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's position during surgery.
2. 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.
3. 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.
4. 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.
5. 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.
6. Free the bone from the muscles attached to it using the periosteal elevator.
7. Inject local anesthesia (0.2 mL of 1% lignocaine) in and around the periosteum to prevent vasovagal reflex.
8. 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.
9. 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.
10. 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.
11. Bend the tip of the K-wire to around 90° 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.
12. 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.
13. Clean the wound with betadine, and cover it with sterile gauze and micropore adhesive tape.

3. Postoperative care

1. 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.
2. 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.
3. Assess bone healing by X-ray of the fractured site once weekly.

4. Radiological procedure

1. 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).
2. 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 ≈ 62; Ref. mAS = 6.4; and automatic exposure settings (Ref. mA=160).
   ​NOTE: X-rays were taken at baseline (1 day after surgery) and then once weekly until sacrifice or 5 weeks.

5. Animal euthanasia and callus retrieval

1. 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.
2. Incise the skin parallel to the femur and separate the overlying muscles carefully to avoid damage to the callus tissue.
3. 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.
4. 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).

6. Decalcification of bone and callus tissue

1. Take the callus tissue from formalin and keep it at RT in 20% ETDA solution, pH 7, for decalcification of the bone tissue.
2. 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.
3. After complete decalcification, cut the sagittal section of the callus and prepare paraffin blocks of the callus tissue. Cut 4 µm thick sections of the callus tissue for histopathological¹⁶ and any other comparative analysis¹⁷.

Results

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...

Discussion

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...

Materials

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