This work was supported by grants from National Natural Science Foundation of China (No. 81772368), Natural Science Foundation of Guangdong Province (No. 2017A030313496), and Guangdong Provincial Science and Technology Plan Project (No. 2016A020215225; No. 2017B090912007). The authors thank Dr. Fei Zhang, M.D. from the Department of Orthopaedic Surgery, The Eighth Affiliated Hospital of Sun Yat-sen University for his technical assistance during modification.

All procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by The Third Affiliated Hospital of Sun Yat-sen University institutional animal care and use committee (permission number: 02-165-01). All the animal experiments were performed according to the ARRIVE guidelines.
1. Preoperative preparation
NOTE: Figure 1 shows the design of the surgical procedure.
<ol>
	<li>Rigidly immobilize the knee joint with a plastic plate and two metal screws at approximately 135&#176; flexion. 
	NOTE: Perform the surgery at the proximal femur and the distal tibia without violating the joint component.</li>
	<li>Prepare materials and instruments for internal fixation.
	<ol>
		<li>Construct a medical grade polypropylene plastic plates by cutting a 5 mL syringe (Figure 2a) using a surgical scissor to fit the following dimensions: length, 25 mm; width, 10 mm; thickness, 1 mm (Figure 2b). Smooth the perimeter of the plate with a scalpel vertically. Rinse the plate with sterile saline to wash off the debris by three times.
		<ol>
			<li>Sterilize with 75% ethanol for 4 h followed by irradiating with ultraviolet light for 3 h.</li>
		</ol>
		</li>
		<li>Pre-drilling holes in the plastic plate: Prepare a hand-held low-speed electric drill with a speed of about 0-4000 rpm (Figure 2c). Drill two holes at both ends of the plate, diameters are 1 mm and 0.9 mm, respectively (Figure 2d). Match both ends of the plate with M 1.4 mm x 8 mm and M 1.2 mm x 6 mm steel screws, respectively (Figure 2e).
		<ol>
			<li>Wipe with 75% ethanol and sterilize with UV light for 3 h before use.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare surgical instruments: 1 straight Mosquito-Type&#8194;hemostatic clamp, 1 smooth curved forceps, 2 eyelid retractors, 1 needle-holder, 1 tissue&#8194;forceps, 1 suture&#8194;scissor, 1 micro tissue&#8194;scissor and 1 scalpel (Figure 2f). Sterilize the surgical instruments by autoclaving at 121.3 &#176;C for 20 min and drying.</li>
	<li>Experimental animals
	<ol>
		<li>Use Specific Pathogen Free (SPF) grade skeletally mature male Sprague-Dawley (or Wistar) rats, weighing between 250 - 350 g in the experiment. 
		NOTE: Choose either female or male rats for the experiment.</li>
		<li>Place the rats in cages and keep in a 12 h light/12 h dark cycle-controlled laboratory room. Provide adequate food and water.</li>
	</ol>
	</li>
</ol>
2. Surgery
<ol>
	<li>Adjust the temperature. Place a warming pad on a surgical platform in a thermostatic operating room.</li>
	<li>Anesthesia and skin preparation
	<ol>
		<li>Weigh the rat with an electronic scale and record.</li>
		<li>Restrain the rat and perform an intraperitoneal injection of sodium pentobarbital (30 mg/kg) to induced anesthesia. Assessing that the animal is sufficiently anesthetized using the toe pinch22. Administer the eyes with lubricant to protect the cornea from drying during surgery.</li>
		<li>Shave the lower body of the rat including the two hind limbs with an electric clipper and disinfect with a tincture of povidone iodine twice and 75% ethanol three times.</li>
		<li>Place the rat laterally, and cover with the surgical drape exposing one side hind leg and hip.</li>
		<li>Disinfect the surgical area again with povidone iodine.</li>
	</ol>
	</li>
	<li>Immobilize the knee joint with internal fixation using a mini-invasive technique. 
	NOTE: Keep the incision properly moist with sterile saline during the operation. The surgery usually requires two surgeons.
	<ol>
		<li>Mark the direction of skin incision. At the distal end of the femur greater trochanter, draw a line along the body surface projection of the muscle gap between the vastus lateralis and biceps femoris (Figure 3a). Incise the epidermis skin along the drawing line approximate 1.5 cm (Figure 3b).</li>
		<li>Bluntly dissect the muscle gap between vastus lateralis and biceps femoris with a tissue forceps until the femoral shaft is exposed approximately 1 cm in length (Figure 3c). Use the retractor to facilitate continuous separation of the muscle gap.</li>
		<li>Incise the epidermis skin approximate 1 cm along the body surface projection of the muscle gap between the tibialis anterior and fibularis longus on the distal lower extremity (Figure 3d). Bluntly dissect the muscle gap until the tibia is exposed approximately 1 cm in length (Figure 3e).</li>
		<li>Separate the soft tissues by the retractor and the smooth forceps, keep perpendicular and drill one 1.0 mm diameter hole into the femoral shaft at a speed of 1,500 rpm using an electric drill (Figure 3f). The proper drilling position is approximate 8 mm below the lower edge of the greater trochanter. Quickly press the wound to stop bleeding. 
		NOTE: Proper drilling diameter can avoid intraoperative fractures.</li>
		<li>Drill one 0.9 mm diameter hole into the tibia approximate 4 mm below the edge of the tibiofibular fusion (Figure 4a). Perform the drilling carefully to prevent the crushing of muscles or tendons.</li>
		<li>Use the straight Mosquito-Type&#8194;hemostatic clamp to form a submuscular course from the tibia hole to femur hole. The submuscular tunnel passes below the gastrocnemius in the tibia end and above the gluteus medius, below the biceps femoris in the femur end.</li>
		<li>Use one M 1.4 mm x 8 mm steel screw to secure one end of the plastic plate (with the 1.0 mm diameter hole) in the proximal femur (Figure 4b). Use one M 1.2 mm x 6 mm steel screw to secure another end of the plastic plate (with the 0.9 mm diameter hole) in the distal tibia (Figure 4c). Ensure the knee joint without varus deformity.</li>
	</ol>
	</li>
	<li>Close the wound: Suture the myofascia, deep fasciae, and subcutaneous tissue using 4-0 absorbable sutures (Figure 4d). Close the skin with Polyamide&#160;sutures (Figure 4f).</li>
</ol>
3. Postoperative management
<ol>
	<li>Apply postoperative analgesia through subcutaneous injection of Buprenorphine (0.03 mg/mL) at 0.05 mg/kg . Add 5 mg/mL Neomycin into drinking water for 5 days after the surgery.</li>
	<li>Inject the analgesia mixture (Buprenorphine and Carprofen) respectively at 0.05mg/kg and 5 mg/kg subcutaneously twice a day for at least 72 hours post-operation.</li>
	<li>Check whether the hind limb had over-edema in case of vascular injury. Made sure that the rats can walk normally in the case of nerve injury during surgery.</li>
</ol>
4. Postoperative examination
<ol>
	<li>Observe the healing of the surgical incision and physically examine the knee joint to evaluate early signs of infection every other day postoperatively. Check the degree of swelling of the ankle and metacarpophalangeal joint in case of continuous edema. 
	NOTE: Early postoperative infection can cause wound exudate, leg swelling, and delayed wound healing.</li>
	<li>Perform X-ray imaging of the hindlimb to ensure that correctly placed the screws on the first postoperative day. 
	NOTE: A Micro-CT scan analysis is another alternative option to display the proper location and the direction of the steel screws.</li>
	<li>Measure the passive range of motion (ROM) to evaluate the development of contracture. Take a knee joint ROM measurement at different time cohorts postoperatively as described previously20.
	<ol>
		<li>In brief, euthanize the rats and skin the hindlimbs. Remove the immobilizer and measure the knee joint angle using a mechanical arthrometer at two torques (667 or 1,060 g/cm)23.</li>
		<li>Calculate the ROM as a result of the total contracture, the myogenic contracture, and the arthrogenic contracture separately based on the investigation objectives24. 
		NOTE: Set different time cohorts (i.e., 1, 2, 4, 8, 16, and 32 weeks) according to the research objectives. The contralateral knee joint (non-operative or sham-operated) can serve as a control2.</li>
	</ol>
	</li>
	<li>Histological analysis of the posterior knee joint capsules.
	<ol>
		<li>Prepare the joint tissues. Dissect the knee joint tissue and fix it with 4% paraformaldehyde. Decalcify and embed it in paraffin as previously reported25. Cut the sections (5 &#956;m) at the medial midcondylar level in the sagittal plane. 
		NOTE: Choose to perform different evaluating staining including HE, aldehyde-fuchsin-Masson Goldner (AFMG), Elastica&#8211;Masson, or Immunohistochemistry staining for histological study in the joint capsule based on your study objectives15,26.</li>
		<li>Observe histomorphometric changes in the posterior knee joint capsules. Photograph the posterior region of the knee joint. Observe fibrous deposition and adhesion changes between the diaphysis-synovium junction and the meniscus6. 
		NOTE: Pathological changes of joint capsule are considered to be a pathogenic factor for knee joint contracture. Measure the length, the thickness, and the capsular areas of the posterior capsule as previously described according to the research content27.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This study aimed to elucidate a step-by-step knee joint immobilization method using a mini-invasive technique that permits rapid postoperative rehabilitation in animals after surgery. Conventionally, the muscle-gap separation approach is thought to be a minimally invasive technique in orthopedic surgery. As expected, we found that rats can return to a normal diet and activities just one day postoperatively, which was consistent with the previous study. Moreover, no artery or nerve injury occurred after the surgery, evidence that the muscle-gap separation modus ensured an adequate and safe bone exposure method. Although the invasive surgical effects can be reduced by using plaster casts, the possibility of edema occurrence in the hind limbs may affect the continuity of immobility. In this study, the ankle or toe swelling caused by surgical procedures disappeared entirely after two days postoperatively. These results highlight a reliable and stable joint immobilization model created by a mini-invasive technique aligned with the principle of rapid recovery. Clinically, the flexion contracture that is caused by immobilization is closer to a non-inflammatory course6. Edema can lead to the release of inflammatory mediators4. Therefore, using plaster casts to induced joint contracture cannot indeed be harmless. In the present study, two separate small incisions (of 1-1.5 cm) were performed on the femoral and tibial sides, respectively. The incision lengths were similar to the size of the incision that is required for K-wire drilling. Therefore, the mini-invasive effect of this method is more conducive to reducing trauma to that of external fixation. Besides, a previous randomized controlled trial demonstrated a possible correlation between the application of external fixation (percutaneously) and the increased risk of infection in the limb16. Considering there no rats had an early infection sign in the research, we assumed that the muscle gap separation technique is the key to this model because it can reduce bleeding and unnecessary cutting. Also, the internal fixator was trimmed down from the syringe, it is low cost and most importantly, non-toxic to animals. Although both the lateral and medial surgical approaches can establish an effective rat model of knee flexion contracture28, this small-invasive technique, however, may only be implemented using the lateral approach rather than using the medial approach.
To our best knowledge, the precise screw drilling position at the proximal femur or distal tibia is not fully understood. Choosing to drill a hole in the middle section of the tibia may affect the blood supply in the tibia. The results obtained from the micro-CT analysis indicated that the proper drilling position is approximate 8 mm below the lower edge of the greater trochanter and approximate 4 mm below the edge of the tibiofibular fusion. The proper drilling position can help avoid effects on the joint component or blood supply. However, the implantation of the internal fixation through a subcutaneous or submuscular way is still controversial. Interestingly, performing the muscle-gap separation technique is convenient for placing the implantation through a submuscular channel to a certain extent.
The results from the joint angle measurement were consistent with the histological analysis, demonstrating that knee joint contracture was successfully induced in the immobilized hindlimb. The average arthrogenic deficits in extension ROM was 29.4 &#177; 3.3&#176;, 40.7 &#177; 4.3&#176; on the immobilized knee joint at the end of 28 days and 56 days of immobilization, respectively, which were significantly higher than that in control (P &#60; 0.05). We also found that typical adhesion had developed between in the joint space between the postero-superior joint capsule and the femur in the immobilized side knee joint (Figure 8e,f), which indicates that using the mini-invasive technique will not interfere with the occurrence of joint contracture. Taken together, the research indicates that this mini-invasive model produces stable results and is effective in inducing acquired joint flexion contracture.
This mini-invasive model still has some limitations. First, the tibia side screw will inevitably irritate the nearby tendons, including the fibularis longus. Second, drilling into the cortical bone may cause fractures. Third, there is still a chance of fixation failure. We believe that the use of 3D-built individualized splints is a possible option for building a non-invasive knee joint contracture model in the future29.
In conclusion, the present study describes a mini-invasive knee joint contracture model that is based on a combination of the muscle gap separation modus and the mini-incision method. Given that internal surgical fixations can produce a well-accepted model of joint contracture, this mini-invasive technique may be useful in the study of immobilization-induced knee flexion contracture.

Joint contractures are defined as a restriction in the passive range of motion (ROM) of a diarthrodial joint1,2. The current therapies aiming to prevent and treat joint contracture have achieved some success3,4. However, the underlying molecular mechanism of acquired joint contracture remains largely unknown5. The etiology of joint contractures in different social communities is highly diverse and includes genetic factors, posttraumatic states, chronic diseases, and prolonged immobility6. It is widely accepted that immobility is a critical issue in the development of acquired joint contracture7. People who suffer from major joint contracture may ultimately result in physical disability8. Thus, a stable and reproducible animal model is necessary for investigating the potential pathophysiological mechanisms of acquired joint contracture.
The currently built immobilization-induced knee joint contracture models are mostly achieved by utilizing non-invasive plaster casts, external fixations, and internal fixations. Watanabe et al. reported the possibility of the use of plaster cast immobilization on rat knee joints9. By wearing a special jacket, one side of the lower limb joint of the rat is immobilized by a cast. The rat knee joint can remain fully flexed without any surgical trauma10,11. However, both the hip and ankle joint movements are also affected by this form of immobilization, which may increase the degree of muscle atrophy in quadriceps femoris or gastrocnemius12. In addition, edema and congestion of the hind limbs must be avoided by replacing the cast at set time points, which may affect the continuity of immobility. Another accepted method for the establishment of a knee joint contracture model is using external surgical fixation. Nagai et al. combined Kirschner wire and steel wire into an external fixator, which immobilized the knee joint to approximately 140&#176; of flexion13. In this method, a resin is used to cover the surface to prevent skin scratches. Although external fixation immobilization is robust and reliable14,15, percutaneous Kirschner wire pin tracks may increase the risk of infection16. In our own experience, using the external fixation technique may reduce the daily activity of rats due to an increase in the conditioned lick behavior.
Alternatively, Trudel et al. described a well-accepted model of joint contracture in the rat knee joint based on a surgical internal fixation17 (this method was modified from the one used by Evans and colleagues18). Notably, this method highlights the importance of utilizing a mini-incision technique to minimize the surgical wounds. The efficient development of joint contracture has been proved in this model19. However, the protocol on how to perform a minimal dissection to expose the bone surface is still unclear20. Also, the precise position where the screw is drilling is not fully understood. The implantation of the internal fixation through a subcutaneous or submuscular way is still controversial21. To solve these problems, we have modified this method by including an appropriate muscle-gap separation modus, which allows a mini-invasive exposure of the bone surface and the placement of the implantation through a submuscular channel. This protocol led to rapid postoperative rehabilitation in rats after surgery. Animals developed a limited joint range of motion after joint immobilization, which was consistent with morphological changes of capsular adhesion obtained from the histological analysis. We also describe an exact possible location of the drilled screws as confirmed by X-ray analysis or micro-CT analysis. Thus, this study aimed to describe in detail a minimal-invasive technique in a knee joint contracture model that was established by a muscle-gap separation modus combined with a mini-incision method. We believe that minimally invasive techniques can both reduce animal trauma and effectively mimic the pathological process of joint flexion contracture.

<table><tbody><tr><td>Anerdian</td><td>Shanghai Likang Ltd.</td><td>310173</td><td>antibacterial</td></tr><tr><td>Buprenorphine&amp;nbsp;</td><td>Shanghai Shyndec Pharmaceutical Ltd.</td><td>/</td><td>analgesia&amp;nbsp;</td></tr><tr><td>Carprofen</td><td>MCE</td><td>HY-B1227</td><td>analgesia&amp;nbsp;</td></tr><tr><td>Cross screwdriver</td><td>STANLEY</td><td>PH0*125mm</td><td>tighten the screws</td></tr><tr><td>Electric drill</td><td>WEGO</td><td>185</td><td>drill hole(with stainless steel drill 0.9mm;1.0mm)</td></tr><tr><td>Microsurgical instruments</td><td>RWD</td><td>/</td><td>Orthopaedic surgical instruments for animals</td></tr><tr><td>Neomycin</td><td>Sigma</td><td>N6386</td><td>antibacterial</td></tr><tr><td>Sodium pentobarbital</td><td>Sigma</td><td>P3761</td><td>&amp;nbsp;anaesthetize</td></tr><tr><td>Stainless Steel screws</td><td>WEGO</td><td>m1.4*8; m1.2*6</td><td>screw(part of internal fixation)&amp;nbsp;</td></tr><tr><td>Syringe&amp;nbsp;</td><td>WEGO</td><td>3151474</td><td>use for plastic plate(part of internal fixation)&amp;nbsp;</td></tr><tr><td>&amp;mu;-CT&amp;nbsp;</td><td>ALOKA</td><td>Latheta LCT-200</td><td>in vivo CT scan</td></tr></tbody></table>

a mini-invasive internal fixation technique for studying immobilization-induced knee flexion contracture in rats

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to restrict knee joint mobility is a widely accepted model to generate experimental contracture. However, implanting application will inevitably cause surgical trauma to the animals. Aiming to develop a less invasive approach, we combined a muscle-gap separation modus with a previously reported mini-incision skill during the surgical procedure: Two mini skin incisions were made on the lateral thigh and leg, followed by performing muscle-gap separation to expose the bone surface. The rat knee joint was gradually immobilized by a preconstructed internal fixation at approximately 135&#176; knee flexion without interfering essential nerves or blood vessels. As expected, this simple technique permits rapid postoperative rehabilitation in animals. The correct position of the internal fixation was confirmed by an x-ray or micro-CT scanning analysis. The range of motion was significantly restricted in the immobilized knee joint than that observed in the contralateral knee joint demonstrating the effectiveness of this model. Besides, histological analysis revealed the development of fibrous deposition and adhesion in the posterior-superior knee joint capsule over time. Thus, this mini-invasive model may be suitable for mimicking the development of immobilized knee joint contracture.

We observed that rats received minimally invasive surgery can return to the regular diet just one day postoperatively. In particular, the surgical incision has scarred without exudate (Figure 5a). The swelling of the ankle and metacarpophalangeal joints in the operative hindlimb has almost wholly disappeared two days postoperatively (Figure 5b) when compared with the contralateral side (Figure 5c). None of the signs of early infection were found in the rats. Rats can stand and exercise regularly (Figure 5d). The surgical wounds had healed entirely on day twelve postoperatively (Figure 5).
Visually, the immobilized knee joint was contracted after four weeks of immobilization, while the mini-invasive surgery had no visible effect on the contralateral limb (Figure 6a). The X-ray image shows the correct placement of the steel screws in the femur or the tibia (Figure 6b), although it did not show the location of the plastic plate. We also employed a high-resolution micro-CT scanner to image the immobilized lower limb. The 3D reconstruction analysis demonstrated that the screws were drilled laterally (Figure 6c). The drilling position is approximate 8 mm below the lower edge of the greater trochanter at the proximal femur and just (approximate 4 mm) below the edge of the tibiofibular fusion at the distal tibia (Figure 6c).
We measured six rats at the end of two times (28 days and 56 days), respectively, to compare the arthrogenic ROM deficits on the immobilized knee joint and the contralateral side after myotomies of the transarticular muscles20. The contralateral knee joint (non-operative) serves as a control. After 28 days of immobilization, the average arthrogenic deficits in extension ROM was 29.4 &#177; 3.3&#176; for the immobilized knee joint, significantly higher than that in control (4.8 &#177; 2.8&#176;, P&#60; 0.05). The arthrogenic deficits in ROM increased during immobilization in a time-dependent manner, demonstrated by the average arthrogenic deficits of 40.7 &#177; 4.3&#176; for the immobilized knee joint, significantly greater than that in control, 11.2 &#177; 3.8&#176; on the 56 days of immobilization (p &#60; 0.05) (Figure 7).
Using Elastica-Masson-Staining, we analyzed the posterior-superior knee joint capsule at three-time points. On day one immobilization, no adhesion was observed in the joint space between the postero-superior joint capsule and the femur in the immobilized or the contralateral side knee joint (Figure 8a,d). However, we observed that there was fibro-adipose tissue deposited and adhesion had developed in the joint space after 28 days of immobilization (Figure 8e). The fibrous tissues even partially replaced this deposition after 56 days of immobilization (Figure 8f) while this type of adhesion was not observed in the contralateral side at different time points (Figure 8 a,b,c).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59260/59260fig1.jpg" /> 
Figure 1: Graphical illustration of a lateral view of the knee joint immobilized with an internal fixation at 135&#176; of flexion. <a href="https://www.jove.com/files/ftp_upload/59260/59260fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59260/59260fig2.jpg" /> 
Figure 2: Design the polypropylene plastic plate into an internal fixation. (a-b) A polypropylene plastic plate was cleaved from the syringe. The dotted lines represent the approximate plate range. The plate has the following dimensions: length, 25 mm; width, 10 mm; thickness, 1 mm. &#160;(c) Photograph of the hand-held electric drill. (d) Drills with the 0.9 mm and 1.0 mm diameter at each end of the plate. The specification of the screw is 1.4 x 8 mm and 1.2 x 6 mm respectively. (e) The final form of a preconstructed internal fixation. (f) The surgical instruments. <a href="https://www.jove.com/files/ftp_upload/59260/59260fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59260/59260fig3.jpg" /> 
Figure 3: Macrographs of surgical exposure the middle femur and the distal tibia using the mini-invasive technique. (a) A black line indicates the skin incision between the vastus lateralis (upper marked area) and biceps femoris (lower marked area). The dotted lines represent the approximate muscle range. (b) The surgical incision between the muscles is illustrated. The incision is away from the sciatic nerve. The black line represents the orientation of the sciatic nerve. (c) The exposure of the femoral midshaft by muscle-gap separation with the vastus lateralis and capput vertebralis indicated. (d-e) The exposure of the tibia is shown in relation to the fibularis longus. (f) The drill hole in the femoral shaft is illustrated with the vastus lateralis, and capput vertebralis indicated. <a href="https://www.jove.com/files/ftp_upload/59260/59260fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59260/59260fig4.jpg" /> 
Figure 4: Implantation of internal fixation. (a) The hole made in the tibia is illustrated with the fibularis longus, and the flexor digitorum profundis indicated. (b-c) The plastic plate screwed into the drill hole is illustrated in relation to the caput vertebralis (b) and the fibularis longus (c). (d-e) Wound closure using vicryl suture. The dotted line (e) represents the approximate plastic plate range. (f) Postoperative overall view of the mini-incision. <a href="https://www.jove.com/files/ftp_upload/59260/59260fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59260/59260fig5.jpg" /> 
Figure 5: Observation of surgical incision healing. (a) The surgical incision has scarred two days postoperatively. (b-c) The swelling of the ankle and metacarpophalangeal joints in the postsurgical limb (b) has almost completely disappeared two days postoperatively. Arrowheads indicate the ankle joints. (d) A rat can stand normally. (e-f) The wound has completely healed twelve days postoperatively. Black arrows indicate surgical healing incision. <a href="https://www.jove.com/files/ftp_upload/59260/59260fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59260/59260fig6.jpg" /> 
Figure 6: Evaluation of knee joint immobilization. (a) The macroscopic image illustrates a contraction of the left knee joint after four weeks of immobilization. (b) Overall x-ray image shows the placement of the screws. (c) Microcomputed tomography analysis of the immobilized knee joint. The white arrows represent the fixed screws. <a href="https://www.jove.com/files/ftp_upload/59260/59260fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59260/59260fig7.jpg" /> 
Figure 7: Analysis of arthrogenic deficits in joint extension range of motion (ROM). Data are presented as mean &#177; SEM (n = 6 per group). The arthrogenic deficits in extension ROM of the immobilized knee joints are significantly higher than that of the contralateral, nonoperative side (serve as a control group). Limitation in ROM represents joint immobilization induced a typical knee flexion contracture. Statistical analysis: The Equality of Variances was performed using Levene&#39;s Test, ROM differences between the contralateral and immobilized groups were compared at two-time point (28 and 56 days) by two tails Student&#8217;s t test. Significance difference was determined by *P &#60; 0.05 from the control. <a href="https://www.jove.com/files/ftp_upload/59260/59260fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/59260/59260fig8.jpg" /> 
Figure 8: Histological changes in the posterior-superior knee joint capsule analyzed by Elastica-Masson-Staining at different time points. Representative images of the posterior-superior joint capsule in the contralateral knee joint (non-operative, upper panels), and the immobilized knee joint (operative, lower panels) on day 1, 28, and 56 during joint immobilization. After a day of immobilization, synovium was thick, and no adhesion was observed in the joint space between the postero-superior joint capsule and the femur (indicated by asterisks in a left row). After 28 days of immobilization, there was fibro-adipose tissue deposited in the joint space and adhesion had developed between postero-superior joint capsule and the femur (indicated by arrowhead). On days 56 of immobilization, the deposits still existed, and there was fibrous tissue increasingly appeared (indicated by arrow). The black border in the bottom left corner represents the magnified image of the joint space between the postero-superior joint capsule and the femur. F: femur; T: tibia; M: meniscus, the posterior horn; JS: joint space. Scale bar = 50 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59260/59260fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats at JoVE.com

A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats

Here, we present a protocol to describe a minimally invasive technique for knee joint immobilization in a rat model. This reproducible protocol, basing on muscle-gap separation modus and the mini-incision skill, is suitable for studying the underlying molecular mechanism of acquired joint contracture.

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to ...

a-mini-invasive-internal-fixation-technique-for-studying

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.9K Views.  The Third Affiliated Hospital of Sun Yat-sen University. Deze eenvoudige en effectieve spier-gap scheiding model techniek combineren de manieren van mini-invasie methode is de beste interne fixatie in onze rat knie gewricht contractie model. Het belangrijkste voordeel van deze techniek is dat het proces reproduceerbaar is en het minimaal invasieve alternatief voor het induceren van contractuur van het kniegewricht bij dieren. Het aantonen van de procedure zal Shihai Jiang en Fei Zhang, afgestudeerde student van mijn laboratorium.Na bevestigi...

Video: A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Medicine

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7, after application of bioactive molecules 8,9, after altering physiological parameters of healing 10, after modifying the amount or time of stabilization 11, after distraction osteogenesis 12, after creation of a non-union 13, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.

This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators 1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role ...

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 ...

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) ...

Bioengineering

Rat Model of Adhesive Capsulitis of the Shoulder

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other etiologies of comparable arthrofibrosis. The model includes extra-articular fixation of the shoulder in rats via scapular to humeral suturing, resulting in a secondary contracture without invading the intra-articular space and resulting in decreased rotational ROM and increased joint stiffness.
We used 10 Sprague-Dawley rats for the purpose of this study. Baseline ROM measurements were taken before glenohumeral immobilization. The rats were subjected to 8 weeks of immobilization before the fixation sutures were removed and changes in ROM and joint stiffness were evaluated. To evaluate whether immobilization resulted in a significant reduction in ROM, changes in kinematics were calculated. ROM was measured at each time point in the follow-up period and was compared to the baseline internal and external ROM measurements. In order to evaluate the stiffness, joint kinetics were calculated by determining the differences in torque (text and tint ) needed to reach the initial external rotation of 60&#176; and initial internal rotation of 80&#176;.
After the removal of the extra-articular suture fixation on follow-up day 0, we found a 63% decrease in total ROM compared to baseline. We observed continuous improvement until week 5 of follow-up, with the progress slowing down around a 19% restriction. On week 8 of follow-up, there was still an 18% restriction of ROM. Additionally, on follow-up day 0, we found the torque increased by 13.3 Nmm when compared to baseline. On week 8, the total torque was measured to be 1.4 &#177; 0.2 Nmm higher than initial measurements. This work introduces a rat model of shoulder adhesive capsulitis with lasting reduced ROM and increased stiffness.

This protocol presents an in vivo rat model of adhesive capsulitis. The model includes an internal fixation of the glenohumeral joint with extra-articular suture fixation for an extended time, resulting in a decreased rotational range of motion (ROM) and increased joint stiffness.

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other ...

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. ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

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.

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. ...

A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats

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