Name: rat model of adhesive capsulitis of the shoulder
Uploaded: 2018-09-28T14:00:00.000+00:00
Duration: 4 min 46 s
Description: Watch this Scientific Journal Video about Rat Model of Adhesive Capsulitis of the Shoulder at JoVE.com

The Authors would like to acknowledge Mr. and Mrs. Tom and Phyllis Froeschle for providing financial support towards this project.

The study was approved by the Institutional Animal Care and Use Committee at Beth Israel Deaconess Medical Center. Care was taken to avoid unnecessary prolonged anesthesia and also to avoid hypothermia. Animals were weighted at each ROM measurement session and monitored for weight loss.
1. Study Subjects
<ol>
	<li>Use 10 Sprague-Dawley rats that are 13 weeks old at the time of surgery and that range between 250&#8211;300 g of body weight.</li>
</ol>
2. Surgical Procedure
<ol>
	<li>Under anesthesia and before surgical immobilization, measure the baseline torque as a function of rotation angle between 60&#176; of external rotation and 80&#176; of internal rotation (see Step 5).</li>
	<li>Induce anesthesia with 5% isoflurane through inhalation in an induction chamber, and then sustain with 2% isoflurane through a nose cone throughout the surgery.
	<ol>
		<li>Use a water-based heating element underneath the animal to maintain body temperature during anesthesia.</li>
		<li>For the purpose of pain control, administer sustained-release buprenorphine subcutaneously at a dose of 1.2 mg/kg depending on the rat&#8217;s body weight.</li>
	</ol>
	</li>
	<li>Immobilize the left glenohumeral joints at an angle of 60&#176; of abduction (the angle between the humeral shaft and the scapular spine)10,11.
	<ol>
		<li>Start the surgical procedure with a posterior longitudinal incision, parallel to the humeral shaft. Make the skin incision just below the glenohumeral joint and extend for about 3 cm.</li>
		<li>Use 2 sutures (braided polyester) to immobilize the glenohumeral joint, by piercing through the lateral edge of the scapula and around the distal two thirds of the humeral shaft, and subsequently tighten as illustrated in Figure 1A. Take extra effort to avoid constricting critical structures such as the brachial artery. 
		Note: The surgical technique has an advantage of being able to limit the glenohumeral joint at 60&#176; of abduction (Z) without affecting other plans (X &#38; Y). By placing sutures around the humerus and through the lateral border of the scapula, an approximation of the two structures is achieved and maintained at the scapular blade plane. After tightening the sutures, the arm is fixed at the resting position when considering X &#38; Y planes and at 60&#176; of abduction when Z plane is considered. This is thought to limit variability between the animals which might be generated from failure of fine tuning the joint position in the three plans.</li>
	</ol>
	</li>
</ol>
3. Closing the Incision
<ol>
	<li>After proper hemostasis, close the skin incision using skin clips.</li>
	<li>Discontinue the anesthesia and allow the animal to recover under supervision in a warm environment. After the animal regains sufficient consciousness to maintain sternal recumbency, return it back to its cage.</li>
	<li>Immediately after the procedure, allow the animals to return to normal activity. Allow the animals to move without restriction, relying mainly on internal sutures to fix the glenohumeral joint.</li>
	<li>To monitor for possible infection, inspect the incision sites daily during the first post-operative week.</li>
	<li>Remove wound clips on the 10th day after surgery. 
	NOTE: There was no manipulation of muscles during the procedure, and the technique did not involve any intra-articular trauma, thus preserving capsular and articular continuity and anatomic integrity. Neither external restrain nor activity limitation was followed in our protocol.</li>
	<li>Provide analgesia using sustained-release buprenorphine, injected subcutaneously at a dose of 1.2 mg/kg starting at anesthesia induction and repeated every 72 h if needed.</li>
</ol>
4. Suture Removal 8 Weeks After Immobilization
<ol>
	<li>Induce anesthesia with 5% isoflurane through inhalation in an induction chamber, then sustained with 2% isoflurane through a nose cone throughout the surgery. For the purpose of pain control, administer sustained-release buprenorphine subcutaneously at a dose of 1.2 mg/kg depending on the rat&#8217;s body weight.</li>
	<li>Make an incision on top of the scar from the previous procedure.</li>
	<li>Cut the sutures and remove from the humerus and the scapula.</li>
	<li>Close the incision with wound clips.</li>
	<li>Examine the incision site daily during the first week to detect any signs of infection. Monitor pain and distress as well.</li>
	<li>Remove wound clips on the 10th day after surgery.</li>
</ol>
5. Range of Motion and Joint Stiffness Measurements
<ol>
	<li>Measure the ROM and passive shoulder mechanics before and after immobilization using a customized device consisting of an arm clamp, a sensor assembly and a pivoting axle11. This is performed pre-operatively (baseline) and continuously after suture removal.
	<ol>
		<li>Measure ROM immediately after suture removal (follow-up day 0), and subsequently twice a week.</li>
		<li>Reduce measurements to once weekly, upon recording less than 10% change from the previous time point. 
		Note: After testing ROM for weeks, the ROM of each animal did not change drastically between testing time points (less than 10% change). The ROM seemed to plateau at this point, therefore, we felt that decreasing testing frequency from twice a week to once a week was sufficient.</li>
	</ol>
	</li>
	<li>Carry out measurements under anesthesia using isoflurane via precision vaporizer at 5% for induction and 2% for maintenance for the full length of the procedure (approximately 5 minutes), in order to facilitate measurements efficiently. Use a water-based heating element underneath the animal to maintain body temperature during anesthesia.</li>
	<li>Position the animal properly for ROM measurement with the aid of a laser guide. Position the forelimb on the arm clamp in 90&#176; forward flexion, with the sensing axis aligned with the long axis of the humerus. Secure the forelimb by the wrist and elbow as illustrated in Figure 1B.</li>
	<li>Control passive forelimb rotation by a stepper motor to assess ROM and torque in a consistent manner. Control the stepper motor with a microcontroller. Use inputs from the inclinometer, in conjunction with those from a torque sensor to indicate the start and end of the measurements.</li>
	<li>Connect the microcontroller to a computer and control using an in-house developed MATLAB code.</li>
	<li>For the baseline measurements only, cycle the sensor assembly 3 times between 60&#176; of external rotation and 80&#176; of internal rotation to obtain initial torque measurements (external: text and internal: tint) for later comparison.</li>
	<li>For ROM measurements, use the torque values of each animal at their own baseline measurements (text and tint) as preset input variables in the program to detect changes in rotation ROM. Use the baseline measurements as a comparative stopping point for subsequent ROM measurements. Changes were detected with a 0.2&#176; resolution.</li>
	<li>For stiffness measurements, use the original rotation angles of 60&#176; external rotation and 80&#176; internal rotation as preset input in the program in order to detect changes in torque. Changes were detected with a resolution of 0.01 Nmm.</li>
	<li>Weigh animals on the same day as each ROM assessment. Place the animals on scales and record their masses. These data were used as one of the tools to assess animal health status during the study.</li>
	<li>Following recovery, return rats to their cages and monitor for signs of pain or distress. Throughout this testing, no animal is left unattended until it has regained sufficient consciousness to maintain sternal recumbency. 
	NOTE: ROM and torque measurements were achieved with a customized device, previously reported by our group {Villa-Camacho 2015}. The device is a custom-made jig that consists of a shaft rotated by stepper motor and controlled by a custom MATLAB script. A torque sensor and inertial measurement unit are used to capture torque and position data during articulation of the specimen.</li>
</ol>
6. Post-mortem Immunohistologic Analysis
<ol>
	<li>At the end of the 8-week ROM measurement period, euthanize rats with CO2 exposure.</li>
	<li>Dissect both left (immobilized) and right (healthy control) shoulders by disarticulating the humerus from the ulna, and by sectioning the scapula from the clavicle and thoracic cavity.</li>
	<li>Fix the excised shoulders in a solution of 10% neutral buffered formalin for 3 days, followed by decalcification in a solution of ethylenediaminetetraacetic acid (EDTA) 10% at pH 7.4 for another 2 months.</li>
	<li>During this process, place samples on a shaker in a gentle agitation cycle and store at 4 &#176;C. Monitor decalcification of the glenohumeral joint with weekly microcomputed tomography (uCT) scans. 
	NOTE: EDTA is a chelating agent that binds calcium ions from the apatite crystal outer surface, progressively reducing the crystal size12,13,14. This process is very slow and gentle, and it is used to detect specific tissue elements that must be preserved for techniques such as immunohistochemistry (IHC). The rate at which the EDTA decalcifies the specimen is dependent on the pH and concentration of the solution. In terms of pH, it can range from 7 to 7.4, with more alkaline solutions accelerating the decalcification speed. However, solutions with higher pH levels can damage key tissue elements. In addition, the usual EDTA concentration for such experiments lies between 10% to 14%, but it is very important to remember that the active agent becomes depleted once it bonds to calcium, so this requires replacement of the decalcification fluid at least 3 to 4 times per week15.</li>
	<li>Once the decalcification process was concluded, mount the glenohumeral joints in paraffin stacks for histological sectioning. Orient the samples to allow for coronal slices. Slices obtained at 50% depth of the humeral head (at the center, mid-coronally) are shown in Figure 1. Perform immunohistochemical staining using the peroxidase-anti-peroxidase method to denote the presence of fibrotic tissue in the joint.4,9,10,16</li>
	<li>Perform antigen retrieval by microwave irradiation by submerging the slides in a plastic Coplin jar in a sodium citrate buffer solution (10 mM sodium citrate, 0.05% Tween 20, pH 6.0, preheated for 5 min at 95&#8211;100 &#176;C) and placing in a regular microwave oven for 10 minutes at medium power. Allow slides to cool for 30 min at room temperature.</li>
	<li>Perform blocking with goat serum for 30 min, and incubate the specimens with a primary mouse mono-clonal antibody (1:400 dilution) to fibronectin overnight at 4 &#176;C.</li>
	<li>After incubation with the primary antibody, wash specimens twice with PBS (0.5 ug/mL) for 10 minutes; and incubate with a secondary antibody, goat antimouse IgG-peroxidase conjugate (1:400 dilution) for 30 minutes.</li>
	<li>Wash the specimens twice with PBS (2.5 &#181;g/mL) on a shaker for 10 minutes and expose to 3,30-diaminobenzidine tetrahydro-chloride and 30% hydrogen peroxide in the dark for 10 min. Counterstain with Carazzi hematoxylin.</li>
</ol>

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Arthroscopy, histological findings and transmission electron microscopy imaging.</a> Chirurg. 72 (11), 1303-1308 (2001).</li><li>Wang, 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=Risk+factors+in+idiopathic+adhesive+capsulitis:+a+case+control+study.">Risk factors in idiopathic adhesive capsulitis: a case control study.</a> Journal of Shoulder and Elbow Surgery. 22 (7), e24-e29 (2013).</li><li>Milgrom, 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=Risk+factors+for+idiopathic+frozen+shoulder.">Risk factors for idiopathic frozen shoulder.</a> The Israel Medical Association Journal. 10 (5), 361-364 (2008).</li><li>Huang, S. 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E., Luevano-Flores, E. <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+different+decalcifying+protocols+on+Osteopontin+and+Osteocalcin+immunostaining+in+whole+bone+specimens+of+arthritis+rat+model+by+confocal+immunofluorescence.">Assessment of different decalcifying protocols on Osteopontin and Osteocalcin immunostaining in whole bone specimens of arthritis rat model by confocal immunofluorescence.</a> International Journal of Clinical and Experimental Pathology. 6 (10), 1972-1983 (2013).</li><li>Sanjai, 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=Evaluation+and+comparison+of+decalcification+agents+on+the+human+teeth.">Evaluation and comparison of decalcification agents on the human teeth.</a> Journal of Oral and Maxillofacial Pathology. 16 (2), 222-227 (2012).</li><li>Rolls, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Introduction to Decalcification. , (2013).</li><li>Burry, R. 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This study presents a rat model of adhesive capsulitis of the shoulder through internal fixation of the glenohumeral joint. Furthermore, it shows an extended reduction of total ROM for at least 8 weeks after removal of the fixation. In order to calculate the alterations in ROM at different time points, measurements were compared to animal specific baselines. Conversely, Kanno et al.10&#160;used a standardized torque for all of the animals in order to determine ex vivo ROM changes.
In 2008, Sarver et al.17 reported on joint stiffness of the shoulder resulting from non-surgical external fixation. Their study showed a transient increase in joint stiffness after immobilization of injured and treated shoulders, which was resolved by week 8 of follow-up. Yet, in the present study, we did not find a linear relationship between torque and angle, rather a polynomial fit (Figure 3). Furthermore, we only found a statistically significant difference in joint stiffness compared to baseline during assessment of internal rotation, where a 25.7% increase in stiffness persisted after 8 weeks of immobilization.
We used baseline ROM and stiffness measurements for each animal as their own internal control. Given the possible variation between the animals18, using the contralateral shoulder of the same animal as internal control increases internal validity and helps reduce the number of animals required.
One of the limitations of our study is that the apparatus used to measure ROM does not stabilize the scapula. However, a rat&#39;s scapula is more obliquely oriented with even more upward rotation than in humans. Having the rats in supine position should theoretically control for scapular tilting as the scapula rests against the firm plate of the testing jig. A further limitation of the study is that we only assess internal-rotation and external-rotation of the glenohumeral joint. This is partly due to the fact that abduction, flexion, and overhead activities require thorough external fixation or restriction of the scapulothoracic joint during ROM testing, which requires a different system than ours.
The pathogenesis and treatment of adhesive capsulitis continues to be inadequately understood. Regardless of the etiology, it has been shown that it is the contracture of the capsule that causes pain and limits glenohumeral movement1,3,11. Furthermore, it has been suggested that there are inflammatory triggers that result in joint fibrosis, thereby causing contracture10. Although our rat model cannot imitate the initial inflammatory insult of a primary contracture, nevertheless, it adequately replicates the characteristic kinetics of adhesive capsulitis and its pathologic changes10,19. This model invokes a lasting reduction in ROM and increased joint stiffness, allowing for a comprehensive assessment of current and potential therapeutic treatments for shoulder contracture.

Adhesive capsulitis of the shoulder is frequently referred to as frozen shoulder or shoulder contracture. It is characterized by restricted glenohumeral motion and pain, presumably as a result of advanced fibrosis and joint contracture1,2,3. The condition involves fibroblast and myofibroblast cell recruitment with a resultant dense collagen matrix (types I and III) in the joint capsule2,3. There are many possible risk factors for developing a joint contracture, including gender, diabetes mellitus, hyperthyroidism, traumatic injury, and prolonged immobilization4,5,6.
Effective treatment options are lacking and mostly include physical therapy, with intervention in the form of surgical release in extreme cases that have not improved with conservative care. The best treatment method remains undetermined and has been a subject of great interest for years in the medical field7,8. Development of novel therapeutic options will require a reproducible animal model for the condition that does not rely on intra-articular induced trauma. The optimal adhesive capsulitis model should involve the two main characteristics of the disease: contracture of the shoulder capsule and a prolonged reduction in range of motion (ROM). Schollmeier et al.9 described one of the first joint contracture models by using a cast to develop shoulder contracture in canines. They also reported that changes in ROM and intra-articular pressure returned to normal levels after cessation of immobilization9. However, an important limitation mentioned in the study is the variation in limb position between animals because of the use of a cast technique.In order to obtain a more reproducible model, Kanno et al.10 later presented an adhesive capsulitis rat model using rigid internal fixation of the shoulder. However, although they achieved a significant reduction in ROM with their model, they did not state whether these changes were temporary or long lasting. The aim of our study was to create a suitable in vivo shoulder contracture rat model by investigating the effect of prolonged extra-articular glenohumeral joint immobilization on ROM and joint stiffness.

<table><tbody><tr><td>Sprague-Dawley rats</td><td>Charles River Laboratories, Wilmington, MA, USA</td><td></td><td>250-300 g</td></tr><tr><td>&lt;strong&gt;Surgical tool:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Injection needle</td><td></td><td></td><td>BD 1&#039; 30 guage</td></tr><tr><td>Needle holder</td><td></td><td></td><td></td></tr><tr><td>5% isoflurane</td><td></td><td></td><td></td></tr><tr><td>2% isoflurane</td><td></td><td></td><td></td></tr><tr><td>Nose cone</td><td></td><td></td><td></td></tr><tr><td>Skalpel and skalpel holder</td><td></td><td></td><td>No. 11 scalpel</td></tr><tr><td>Curved hemostat forceps</td><td></td><td></td><td></td></tr><tr><td>Staright hemostat forceps</td><td></td><td></td><td></td></tr><tr><td>Tissue retractor</td><td></td><td></td><td></td></tr><tr><td>Toothed tissue forceps</td><td></td><td></td><td></td></tr><tr><td>Plain tissue forceps</td><td></td><td></td><td></td></tr><tr><td>Dissecting scissors</td><td></td><td></td><td></td></tr><tr><td>Suture scissors</td><td></td><td></td><td></td></tr><tr><td>Skin clip applicator</td><td></td><td></td><td>Any standard staples for wound closure</td></tr><tr><td>Immobilization material</td><td>Ethicon</td><td></td><td>No. 2-0 braided polyester ethibond suture was used for immobilization</td></tr><tr><td>&lt;strong&gt;Other materials:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Costumized device for ROM: 1)Sensor assembly, 2)pivoting axle, 3)arm clamp</td><td></td><td></td><td>Assembly that is described in relaxin paper and adhesive capsulitis paper</td></tr><tr><td>Orientation sensor (part of sensor assembly)</td><td>MicroStrain Inc., Williston, VT, USA</td><td></td><td>3DM-GX3-15</td></tr><tr><td>Reaction torque sensor (part of sensor assembly)</td><td>Futek Inc., Irvine, CA, USA</td><td></td><td>TFF400</td></tr><tr><td>Stepper Motor</td><td>SparkFun Electronics, Niwot, CO 80503</td><td></td><td>https://www.sparkfun.com/products/13656</td></tr><tr><td>Microcontroller</td><td>Torino, Italy).</td><td></td><td>Arduino UNO, R3</td></tr><tr><td>MATLAB code</td><td>MATLAB 7.13.0.564, Natick, Ma, USA</td><td></td><td></td></tr><tr><td>Weight Scale</td><td></td><td></td><td>Ohaus</td></tr></tbody></table>

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.

Range of motion
On follow-up day 0, we found a 63% decrease in total ROM compared to baseline (P &#60; .001). We observed a gradual improvement of ROM until week 5 of follow-up, when progression stopped at 19% restriction (P &#60;0.001). The remaining restriction, 18% of total ROM, was still apparent at 8 weeks of follow-up (P &#60;0.001).
Stiffness
On follow-up day 0, we found an increase of 13.3 Nmm in total torque compared with baseline (P &#60;0.001); 8.9 Nmm externally (P = .002) and 4.4 Nmm internally (P &#60;0.001), resulting in a 138.8% increase in external rotation torque, 159.6% increase in internal rotation torque, and a total of 149.2% increased torque overall. On week 8 of follow-up, we found the total measured torque to be 1.4 &#177; 0.2 Nmm higher than baseline (P = 0.115), with a 0.6&#160;&#177; 0.1 Nmm increase of external torque (P = 0.369) and 0.7&#160;&#177; 0.2 Nmm increase of internal torque (P = 0.036). This indicates torque increases 10% externally and 25.7% internally, for an overall 17.9% increase. At the beginning of post-operative week 3, the improvement of stiffness plateaued.
Histologic Results
As seen in Figure 2A, the intact group displayed proper separation between the capsule and articular surface of the femoral head and normal cellular organization. In addition, normal cellular organization was also observed in the synovial tissue and articular cartilage. However, the surgically immobilized group shows evidence of capsular adhesions in the inferior aspect of the glenohumeral joint. Moreover, the surrounding tissue appears to be denser when compared to the intact shoulders, leading to a tighter capsule with a decreased joint space (Figure 2B). Slices stained for fibronectin show an increased capsular thickness in the contracted surgical group (Figure 2B). when compared to healthy controls (Figure 2A). These findings are in agreement with previously reported literature on animal models of joint immobilization10 and support the creation of a proper contracture model.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58335/58335fig1.jpg" /> 
Figure 1: Testing Apparatus. (A) Immobilization of the glenohumeral joint with 2 braided polyester sutures, passed firmly between the lateral edge of scapular and the humerus; (B) Customized device for measurement of ROM and passive shoulder mechanics; a) A stepper monitor. A sensor assembly consisting of b) a reaction torque sensor, and c) an orientation sensor. d) an arm clamp. (C) Internal or (D) external rotation of the glenohumeral joint11. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58335/58335fig1large.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/58335/58335fig2.jpg" /> 
Figure 2: Coronal slices of the humeral head. Image stained for fibronectin (IHC) obtained at 40X magnification. Scale bar = 200 &#181;m. (A) Healthy control. (B) Surgical control. Red arrows outline joint capsular thickness. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58335/58335fig2large.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/58335/58335fig3.jpg" /> 
Figure 3: Range of motion versus normalized torque for both healthy and surgically restricted rat glenohumeral joints. Internal Rotation is denoted as positive, external rotation is negative. The shaded region shows the 95% Confidence Interval (CI). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58335/58335fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Rat Model of Adhesive Capsulitis of the Shoulder at JoVE.com

Rat Model of Adhesive Capsulitis of the Shoulder

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 ...

rat-model-of-adhesive-capsulitis-of-the-shoulder

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

7.3K Views.  Beth Israel Deaconess Medical Center and Harvard Medical School. 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.

Video: Rat Model of Adhesive Capsulitis of the Shoulder

Reverse Total Shoulder Arthroplasty

Reverse total shoulder arthroplasty was initially approved for use in rotator cuff arthropathy and well as chronic pseudoparalysis without arthritis in patients who were not appropriate for tendon transfer reconstructions. Traditional surgical options for these patients were limited and functional results were sub-optimal and at times catastrophic. The use of reverse shoulder arthroplasty has been found to effectively restore these patients function and relieve symptoms associated with their disease. The procedure can be done through two approaches, the deltopectoral or the superolateral. Complication rates associated with the use of the prosthesis have ranged from 8-60% with more recent reports trending lower as experienced is gained. Salvage options for a failed reverse shoulder prosthesis are limited and often have significant associated disability. Indications for the use of this prosthesis continue to be evaluated including its use for revision arthroplasty, proximal humeral fracture and tumor. Careful patient selection is essential because of the significant risks associated with the procedure.

Reverse total shoulder arthroplasty is indicated for the treatment of conditions that cannot be treated with conventional arthroplasty or other procedures. These primarily include degenerative and incapacitating conditions with irreparable rotator cuff and loss of the normal biomechanical coupling of the shoulder.

Reverse total shoulder arthroplasty was initially approved for use in rotator cuff arthropathy and well as chronic pseudoparalysis without arthritis in ...

Medicine

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Measurement of Dynamic Scapular Kinematics Using an Acromion Marker Cluster to Minimize Skin Movement Artifact

The measurement of dynamic scapular kinematics is complex due to the sliding nature of the scapula beneath the skin surface. The aim of the study was to clearly describe the acromion marker cluster (AMC) method of determining scapular kinematics when using a passive marker motion capture system, with consideration for the sources of error which could affect the validity and reliability of measurements. The AMC method involves placing a cluster of markers over the posterior acromion, and through calibration of anatomical landmarks with respect to the marker cluster it is possible to obtain valid measurements of scapular kinematics. The reliability of the method was examined between two days in a group of 15 healthy individuals (aged 19-38 years, eight males) as they performed arm elevation, to 120&#176;, and lowering in the frontal, scapular and sagittal planes. Results showed that between-day reliability was good for upward scapular rotation (Coefficient of Multiple Correlation; CMC = 0.92) and posterior tilt (CMC = 0.70) but fair for internal rotation (CMC = 0.53) during the arm elevation phase. The waveform error was lower for upward rotation (2.7&#176; to 4.4&#176;) and posterior tilt (1.3&#176; to 2.8&#176;), compared to internal rotation (5.4&#176; to 7.3&#176;). The reliability during the lowering phase was comparable to results observed during the elevation phase. If the protocol outlined in this study is adhered to, the AMC provides a reliable measurement of upward rotation and posterior tilt during the elevation and lowering phases of arm movement.

This report presents details of how to adopt the acromion marker cluster method of obtaining scapular kinematics when using a passive marker motion capture device. As has been described in the literature, this method provides a robust, non-invasive, three-dimensional, dynamic and valid measurement of scapular kinematics, minimizing skin movement artifact.

The measurement of dynamic scapular kinematics is complex due to the sliding nature of the scapula beneath the skin surface. The aim of the study was to ...

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, multiple ligaments, and approximately 20 muscles. Unfortunately, shoulder pathologies (e.g., rotator cuff tears, joint dislocations, arthritis) are common, resulting in substantial pain, disability, and decreased quality of life. The specific etiology for many of these pathologic conditions is not fully understood, but it is generally accepted that shoulder pathology is often associated with altered joint motion. Unfortunately, measuring shoulder motion with the necessary level of accuracy to investigate motion-based hypotheses is not trivial. However, radiographic-based motion measurement techniques have provided the advancement necessary to investigate motion-based hypotheses and provide a mechanistic understanding of shoulder function. Thus, the purpose of this article is to describe the approaches for measuring shoulder motion using a custom biplanar videoradiography system. The specific objectives of this article are to describe the protocols to acquire biplanar videoradiographic images of the shoulder complex, acquire CT scans, develop 3D bone models, locate anatomical landmarks, track the position and orientation of the humerus, scapula, and torso from the biplanar radiographic images, and calculate the kinematic outcome measures. In addition, the article will describe special considerations unique to the shoulder when measuring joint kinematics using this approach.

Biplane videoradiography can quantify shoulder kinematics with a high degree of accuracy. The protocol described herein was specifically designed to track the scapula, humerus, and the ribs during planar humeral elevation, and outlines the procedures for data collection, processing, and analysis. Unique considerations for data collection are also described.

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...

Understanding Tendon Impingement &#8211; Murine Explant Model for Studying Mechanobiology

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

Author Spotlight: Unraveling the Mechanobiology of Tendon Impingement &#8211; A Multiaxial Murine Hind Limb Explant Model 

We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement.

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a ...

Treatment Protocol for Rotator Cuff Calcific Tendinitis Using a Single-Crystal Piezoelectric Focused Shock Wave Source

Focused shock waves have emerged as a highly effective noninvasive therapeutic option for the treatment of calcific tendinitis of the shoulder. There are three types of focused shock wave generators: electrohydraulic, electromagnetic, and piezoelectric. According to our literature search, there are no reports of results with the use of single-crystal piezoelectric generators in calcific tendinitis of the shoulder. In a consecutive retrospective series of 23 patients with G&#228;rtner type I and II calcifications of the rotator cuff, we performed three applications of high-energy piezoelectric focused waves (4,000 pulses per session with a frequency of 6 Hz). At the final follow-up (average of 14 months), 82.6% of the cases showed complete resorption of the calcification in radiographic controls. In 8.7% of the cases, partial disappearance of the calcification was achieved, and in the remaining 8.7% there were no significant changes. Single-crystal piezoelectric generators have a success rate comparable to those already reported with electrohydraulic and electromagnetic devices.

Calcific tendinitis of the shoulder is a relatively common condition with numerous treatment options. Here, we discuss the indications of focused shock waves generated by a single-crystal piezoelectric device, describe a treatment protocol, and present the preliminary results.

Focused shock waves have emerged as a highly effective noninvasive therapeutic option for the treatment of calcific tendinitis of the shoulder. There are ...

Tuina Therapy for Frozen Shoulders in a Rat Model

Tuina in a Frozen Shoulder Rat Model: An Efficient and Reproducible Protocol

Frozen shoulder (FS) is a common condition with no defined optimal therapy. Tuina therapy, a traditional Chinese medicine (TCM) technique used to treat FS patients in Chinese hospitals, has demonstrated excellent results, but its mechanisms are not fully understood. Building on a previous study, this work aimed to develop a Tuina protocol for an FS rat model. We randomly divided 20 SD rats into control (C; n = 5), FS model (M; n = 5), FS model Tuina treatment (MT; n = 5), and FS model oral treatment (MO; n = 5) groups. This study used the cast immobilization method to establish the FS rat model. The effect of Tuina and oral dexamethasone on the glenohumeral range of motion (ROM) was evaluated, and the histological findings were assessed. Our study showed that Tuina and oral dexamethasone were able to improve shoulder active ROM and preserve the structure of the capsule, with Tuina therapy proving to be more effective than oral dexamethasone. In conclusion, the Tuina protocol established in this study was highly effective for FS.

Author Spotlight: Investigating the Effectiveness of Tuina Therapy for Frozen Shoulder 

This study develops an efficient and reproducible Tuina protocol for treating frozen shoulder established in a rat model. This approach will help to study the Tuina therapy treatment method for frozen shoulders.

Frozen shoulder (FS) is a common condition with no defined optimal therapy. Tuina therapy, a traditional Chinese medicine (TCM) technique used to treat FS ...

Therapeutic Efficacy of Small Needle Knife Therapy in Treating Frozen Shoulder

Clinical Efficacy of Small Needle Knife Therapy on Stage I-II Frozen Shoulder

Frozen shoulder is a kind of aseptic inflammatory disease of the shoulder caused by strain, trauma, and other reasons, resulting in shoulder joint pain and limited function. The protocol presented here demonstrates the operation of a small needle knife in treating frozen shoulders, including patient management, material preparation, positioning, operation, and postoperative care. The purpose of this protocol is to relieve the pain and functional limitations and improve the living ability of patients with frozen shoulders. In our study, 76 stage I-II frozen shoulder patients who met the inclusion criteria were randomly divided into a control group and a treatment group (n=38). Patients in the control group received functional exercise, while the treatment group received small needle knife therapy with functional exercise. The visual analogue scores (VAS), the Constant and Murley scores (CMS), and the thickness of the coracohumeral ligament (CHL) under ultrasound were evaluated. After small needle knife therapy, the VAS score was significantly lower in the treatment group (5.11 &plusmn; 0.89) than in the control group (5.49 &plusmn; 0.65; t=-2.065, p&lt;0.05); the CMS score was significantly higher in the treatment group (64.72 &plusmn; 4.78) than in the control group (60.97 &plusmn; 6.00; t=2.947, p&lt;0.05); the CHL thickness was significantly decreased in the treatment group (2.38 &plusmn; 0.36) than in the control group (2.57 &plusmn; 0.42; t=-2.117, p&lt;0.05). These results indicate that the small needle knife significantly relieved the pain symptoms, improved the shoulder function, reduced the CHL thickness, and improved the quality of life and, therefore, had significant therapeutic efficacy in stage I-II frozen shoulder patients.

Author Spotlight: Treating Frozen Shoulder with Small Needle Knife Therapy 

The protocol presented here describes the detailed operation method and proves the effectiveness of a small needle knife in the treatment of frozen shoulders.

Frozen shoulder is a kind of aseptic inflammatory disease of the shoulder caused by strain, trauma, and other reasons, resulting in shoulder joint pain ...

Development of a Rabbit Chronic-Like Rotator Cuff Injury Model for Study of Fibrosis and Muscular Fatty Degeneration

Rabbit rotator cuff (RC) pathophysiology can lead to progressive and highly degenerative changes in its associated musculature and tendons, which negatively influences clinically relevant parameters, such as strength and retraction of the muscle-tendon/myotendinous unit, ultimately causing loss of shoulder function and negatively affecting RC repair outcomes. Animal models that mimic aspects of human RC anatomy and pathophysiology are crucial for advancing the conceptual understanding of injury progression and developing effective tissue engineering and regenerative medicine-based therapeutics.
Within this context, a rabbit subscapularis (SSC) model is suitable due to (i) its anatomical similarity to the human supraspinatus (SSP) bone-tendon-muscle unit, which is the most frequently injured RC site; (ii) its pathophysiological similarity to humans in terms of fibrosis and muscle fatty degeneration (FD); and (iii) its amenability to surgical procedures. Therefore, the goal of this study is to describe the surgical techniques for inducing SSC RC injury. Briefly, the procedure involves the isolation of the SSC by identifying the coracobrachialis muscle followed by a full-thickness transection at the muscle-tendon junction and wrapping the free end of the muscle-tendon junction with a silicone-based penrose tubing to prevent spontaneous reattachment. Histologic evaluations are performed to monitor the progression of muscle FD at 4 weeks post-surgery using hematoxylin and eosin (H&#38;E) as well as Masson&#39;s trichrome staining.
Loss of muscle and FD were evident 4 weeks after transection of the SSC muscle-tendon junction, similar to human RC pathophysiological conditions. This protocol demonstrates the steps for successfully establishing a chronic-like rabbit SSC RC injury model, which can serve as a powerful tool to study skeletal muscle changes associated with RC pathophysiology and aid the development of novel therapeutic strategies for chronic-like RC tears.

This study details procedures for establishing a chronic-like rabbit rotator cuff (RC) injury. Specifically, the injury is created in the subscapularis (SSC) muscle-tendon/myotendinous unit to mimic human RC anatomy and pathophysiology, including severe muscle fatty degeneration (FD). This protocol can be applied to study RC injuries and assess regenerative therapies.

Rabbit rotator cuff (RC) pathophysiology can lead to progressive and highly degenerative changes in its associated musculature and tendons, which ...

Biology

Dermal Allograft and Biceps Superior Capsule Reconstruction for Massive Irreparable Rotator Cuff Tears

Since the use of autologous fascia lata for superior capsule reconstruction of massive irreparable rotator cuff tears (MIRCTs), the technique has evolved into various modifications, including dermal allografts, long head of the biceps tendon (LHBT), and combinations of both, which will be discussed in this article. After making sure the remnant cuff cannot be restored to the anatomical footprint of the supraspinatus, a double-loaded or triple-loaded, suture-based anchor is inserted 5-8 mm posterior to the bicipital groove to secure the long head of the biceps (LHBT) initially. A bone trough is made 5 mm posterior to the bicipital groove. One or two lasso loops are created through the LHBT before the complete release of the transverse humeral ligament without tenotomy of the LHBT distal to the fixation point, resulting in a posteriorly rerouted LHBT.Preservation of the proximal attachment of the biceps on the glenoid side is maintained, ensuring a native fixation. 
Subsequently, a 3 x 3 cm dermal allograft of 2 mm thickness is utilized to cover the rerouted LHBT, enhancing its strength and providing a tensile effect. Four anchors are then employed for fixation: two double-loaded anchors at the glenoid side and two lateral row anchors at the greater tuberosity. Following the introduction of the dermal allograft into the joint, the sutures from the glenoid anchors are secured, and the optimal tension of the allograft is gauged during the insertion of the lateral row anchors at 45&#176; shoulder abduction. The dermal allograft can cover the LHBT to increase the spacer effect. Medial row anchors are not necessary. The remaining portions of the supraspinatus and infraspinatus can be repaired using sutures passed through the lateral row anchors or repaired with the dermal allograft together to enhance stability.

Here, we describe a modified technique for dermal allograft and long head of the biceps tendon superior capsule reconstruction for massive irreparable rotator cuff tears.

Since the use of autologous fascia lata for superior capsule reconstruction of massive irreparable rotator cuff tears (MIRCTs), the technique has evolved ...