Name: rat model of adhesive capsulitis of the shoulder
Uploaded: 2018-09-28T14:00:00
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 Procedur...

<ol>
	<li>Bunker, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+for+a+new+name+for+'frozen+shoulder'.">Time for a new name for 'frozen shoulder'.</a> British medical journal. 290 (6477), 1233-1234 (1985).</li><li>Bunker, T. D., Anthony, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathology+of+frozen+shoulder.+A+Dupuytren-like+disease.">The pathology of frozen shoulder. A Dupuytren-like disease.</a> The Journal of bone and joint surgery. British volume. 77 (5), 677-683 (1995).</li><li>Kilian, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+frozen+shoulder.+Arthroscopy,+histological+findings+and+transmission+electron+microscopy+imaging.">The frozen shoulder. 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. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperthyroidism+is+a+risk+factor+for+developing+adhesive+capsulitis+of+the+shoulder:+a+nationwide+longitudinal+population-based+study.">Hyperthyroidism is a risk factor for developing adhesive capsulitis of the shoulder: a nationwide longitudinal population-based study.</a> Scientific Reports. 4, 4183 (2014).</li><li>Struyf, F., Meeus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+evidence+on+physical+therapy+in+patients+with+adhesive+capsulitis:+what+are+we+missing.">Current evidence on physical therapy in patients with adhesive capsulitis: what are we missing.</a> Clinical Rheumatology. 33 (5), 593-600 (2014).</li><li>Song, A., Higgins, L. D., Newman, J., Jain, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glenohumeral+corticosteroid+injections+in+adhesive+capsulitis:+a+systematic+search+and+review.">Glenohumeral corticosteroid injections in adhesive capsulitis: a systematic search and review.</a> Journal Of Physical Medicine And Rehabilitation. 6 (12), 1143-1156 (2014).</li><li>Schollmeier, G., Sarkar, K., Fukuhara, K., Uhthoff, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+changes+in+the+canine+shoulder+after+cessation+of+immobilization.">Structural and functional changes in the canine shoulder after cessation of immobilization.</a> Clinical Orthopaedics and Related Research. 323 (323), 310-315 (1996).</li><li>Kanno, A., Sano, H., Itoi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+shoulder+contracture+model+in+rats.">Development of a shoulder contracture model in rats.</a> Journal of Shoulder and Elbow Surgery. 19 (5), 700-708 (2010).</li><li>Villa-Camacho, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+kinetic+evaluation+of+an+adhesive+capsulitis+model+in+rats.">In vivo kinetic evaluation of an adhesive capsulitis model in rats.</a> Journal of Shoulder and Elbow Surgery. 24 (11), 1809-1816 (2015).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Decalcification+Techniques+for+Rat+Femurs+Using+HE+and+Immunohistochemical+Staining.">Evaluation of Decalcification Techniques for Rat Femurs Using HE and Immunohistochemical Staining.</a> BioMed Research International. 2017, 9050754 (2017).</li><li>Gonzalez-Chavez, S. A., Pacheco-Tena, C., Macias-Vazquez, C. 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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+for+immunocytochemistry:+an+update.">Controls for immunocytochemistry: an update.</a> Journal of Histochemistry &amp; Cytochemistry. 59 (1), 6-12 (2011).</li><li>Sarver, J. J., 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=After+rotator+cuff+repair,+stiffness--but+not+the+loss+in+range+of+motion--increased+transiently+for+immobilized+shoulders+in+a+rat+model.">After rotator cuff repair, stiffness--but not the loss in range of motion--increased transiently for immobilized shoulders in a rat model.</a> Journal of Shoulder and Elbow Surgery. 17 (1 Suppl), 108S-113S (2008).</li><li>. Variations within Outbred Strains: Know Your Strains and Stocks | Taconic Biosciences Available from: <a target="_blank" href="https://www.taconic.com/taconic-insights/quality/variations-within-outbred-strains-know-your-strains-and-stocks.html">https://www.taconic.com/taconic-insights/quality/variations-within-outbred-strains-know-your-strains-and-stocks.html</a> (2016)</li><li>Liu, Y. L., Ao, Y. F., Cui, G. Q., Zhu, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+of+histology+and+capsular+collagen+in+a+rat+shoulder+immobilization+model.">Changes of histology and capsular collagen in a rat shoulder immobilization model.</a> Chinese Medical Journal. 124 (23), 3939-3944 (2011).</li></ol>

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...

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>Surgical tool:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Injection needle</td>
			<td></td>
			<td></td>
			<td>BD 1&#39; 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>Other materials:</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
<p class="jove_conten...

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 ...

This method can help answer key questions in the orthopedic surgery field, such as, Would relaxing to improve the range of motion in patients suffering from arthrofibrosis? The main advantage of this technique is that it avoid the section of shoulder girdle muscles and any intra-articular trauma, thus preserving capsular and articular continuity and anatomic integrity. Before beginning the procedure, connect the microcontroller of a Stepper Motor to a computer and set up an in-house-developed Matlab code to control the microcontroller.Next, position a 13-week-old, 250 to 300 gram Sprague Dawley rat in a customized range of motion device with the aid of a laser guide. Then, position the left forelimb of the rat on the arm clamp in 90 degrees forward flexion with the sensing axis aligned with the long axis of the humerus and secure the forelimb by the wrist and elbow. Then, use the microcontroller to control the passive forelimb rotation and cycle the sensor assembly three times between 60 degrees of external rotation and 80 degrees of internal rotation to obtain baseline torque measurements using inputs from the inclinometer in conjunction with those from a torque sensor to indicate the start and end of the measurements.To perform the surgical immobilization of the shoulder joint, after confirming a lack of response to toe pinch, make a posterior longitudinal incision parallel to the humeral shaft starting just below the shoulder joint and extending for about three centimeters. Using two braided polyester sutures, pierce through the medial edge of the scapula and around the distal two-thirds of the humeral shaft. And subsequently tighten the sutures to immobilize the shoulder joint.Use wound clips to close the incisions and monitor the incision sites for infection daily during the first post-operative week. Eight weeks after the immobilization, make an incision on top of the scar from the previous procedure and remove the sutures from humerus and the scapula. Then close the incision with fresh wound clips and measure the passive shoulder mechanics as demonstrated.To measure the range of motion, use the torque values at baseline as preset input variables in the program to detect changes in the rotation range of motion with a 0.2 degree resolution. For stiffness measurements, use the original rotation angles of 60 degrees external rotation and 80 degrees internal rotation as the preset inputs in the program to detect the changes in torque with a resolution of 0.01 newton millimeters. Weigh the animal on the same day as each range of motion assessment as one of the tools for evaluating animal health status during the study.Then return the animal to its cage with daily monitoring during the first week for signs of infection, pain, or distress, and removing the wound clips on the 10th day after the second surgery. The intact group displays a proper separation between the capsule and the articular surface of the humeral head as well as a normal cellular organization in the synovial tissue and articular cartilage. The surgically immobilized group, however, exhibits evidence of capsular adhesions in the inferior aspect of the shoulder joint.Moreover, the surrounding tissue appears to be denser as compared to the intact shoulders, leading to a tighter capsule with a decreased joint space. While attempting this procedure, it's important to remember to position the animals correctly. Following this procedure, other methods like intraperitoneal injection of Relaxin-2 can be performed to answer additional questions about the systemic effects of Relaxin-2.After its development, this technique paved the way for researchers in the field of orthopedic surgery to explore the use of Relaxin-2 in reversing fibrosis in different joints due to various underlying pathologies. Don't forget that working with animals can be dangerous and that precautions such as proper animal handling and wearing personal protective equipment should always be taken while performing this procedure.

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

Research

JoVE Journal

Bioengineering

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.1 Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.2 Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin 'solders' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.3 Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.4
In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed 'SurgiLux'. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1).
In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.5 Without this 'activation' process, SurgiLux films are readily removed.6-9 SurgiLux has been tested both in vitro and in vivo on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.6-10

The fabrication of a novel, flexible thin film surgical adhesive from FDA approved ingredients, chitosan and indocyanine green is described. Bonding of this adhesive to collagenous tissue through a simple activation process with a low-powered infra-red laser is demonstrated.

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

Microelectrode arrays for neural interface devices that are made of biocompatible thin-film polymer are expected to have extended functional lifetime because the flexible material may minimize adverse tissue response caused by micromotion. However, their flexibility prevents them from being accurately inserted into neural tissue. This article demonstrates a method to temporarily attach a flexible microelectrode probe to a rigid stiffener using biodissolvable polyethylene glycol (PEG) to facilitate precise, surgical insertion of the probe. A unique stiffener design allows for uniform distribution of the PEG adhesive along the length of the probe. Flip-chip bonding, a common tool used in microelectronics packaging, enables accurate and repeatable alignment and attachment of the probe to the stiffener. The probe and stiffener are surgically implanted together, then the PEG is allowed to dissolve so that the stiffener can be extracted leaving the probe in place. Finally, an in vitro test method is used to evaluate stiffener extraction in an agarose gel model of brain tissue. This approach to implantation has proven particularly advantageous for longer flexible probes (&gt;3 mm). It also provides a feasible method to implant dual-sided flexible probes. To date, the technique has been used to obtain various in vivo recording data from the rat cortex.

Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.

Microelectrode  arrays for neural interface devices that are made of biocompatible thin-film  polymer are expected to have extended functional lifetime ...

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 ...

Use of a Rat Model to Study Ventral Abdominal Hernia Repair

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal hernia typically requires implantation of a material to serve as a mechanical bridge across the defect in the abdominal wall. Biomaterials, such as porcine small intestinal submucosa (SIS), also serve as a lattice for cell growth into the implant and can naturally incorporate into the host tissue. Development of such repair materials benefits from use of animal models in which experimental abdominal wall defects are easily created and are amenable to repair in a reproducible fashion. The method offered here describes surgical creation and repair of ventral abdominal hernia in a rat model. When SIS is used to repair an experimental ventral abdominal hernia in this model, it is rapidly incorporated into host tissue within 28 days of implantation. Histologically, incorporation of their implanted material into host tissue is characterized by a robust fibrovascular response. Future refinements and applications of the rat abdominal hernia model may likely involve diabetic and/or obese animals as a means to more closely mimic common co-morbidities of man.

This manuscript describes methods associated with creation and repair of a ventral abdominal wall defect (hernia). This model can be used to study repair strategies such as those that use implanted materials. In this manuscript, repair of the experimental hernia with porcine small intestinal submucosa is presented as an example.

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

Fabrication of the Composite Regenerative Peripheral Nerve Interface (C-RPNI) in the Adult Rat

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is often accomplished through integration with the peripheral nervous system. Unfortunately, methods currently employed are often associated with significant tissue damage which prevents prolonged use. Additionally, these devices often lack any meaningful degree of sensory feedback as their complex construction dampens any vibrations or other sensations a user may have previously depended on when using more simple prosthetics. The composite regenerative peripheral nerve interface (C-RPNI) was developed as a stable, biologic construct with the ability to amplify efferent motor nerve signals while providing simultaneous afferent sensory feedback. The C-RPNI consists of a segment of free dermal and muscle graft secured around a target mixed sensorimotor nerve, with preferential motor nerve reinnervation of the muscle graft and sensory nerve reinnervation of the dermal graft. In rats, this construct has demonstrated the generation of compound muscle action potentials (CMAPs), amplifying the target nerve&#39;s signal from the micro- to milli-volt level, with signal to noise ratios averaging approximately 30-50. Stimulation of the dermal component of the construct generates compound sensory nerve action potentials (CSNAPs) at the proximal nerve. As such, this construct has promising future utility towards the realization of the ideal, intuitive prosthetic.

Fabrication of the Composite Regenerative Peripheral Nerve Interface C-RPNI in the Adult Rat

The following manuscript describes a novel method for developing a biologic, closed loop neural feedback system termed the composite regenerative peripheral nerve interface (C-RPNI). This construct has the ability to integrate with peripheral nerves to amplify efferent motor signals while simultaneously providing afferent sensory feedback.

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

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, ...