The authors wish to thank Valerie Schroeder for her assistance with technical aspects of this work. Work related to development of this model was supported by Cook Biotech, Inc. (West Lafayette, IN USA).

The use of animals in this protocol was approved by the University of Notre Dame Institutional Animal Care and Use Committee, followed all regulatory requirements and guidelines, and was conducted in a facility that is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), International.
1. Selection of Animals
<ol>
	<li>Acquire pathogen-free Sprague Dawley rats from a reputable vendor or breeding colony. 
	Note: Often, the facility veterinarian or manager can direct the investigator to appropriate sources. Rats of approximately 250 g body weight are suitable for this procedure.</li>
	<li>One day prior to surgery, examine the animals to be used to ensure that they are clinically healthy. In this regard, weigh the animal and assess the body condition; note any abnormality in respiration, the presence of any open wounds or masses, and the general activity level of the animal. Note any abnormality in the surgical record and consider such observations as potential exclusion criteria.</li>
</ol>
2. Anesthesia and Preparation of Animal for Aseptic Surgery
<ol>
	<li>For analgesia, administer 1.1 - 1.2 mg/kg of sustained release buprenorphine, subcutaneously to the rat 2 h&#160;before surgery.&#160;Apply ophthalmic ointment to both eyes, and administer 2 mL of warmed sterile physiologic saline subcutaneously.</li>
	<li>Anesthetize the rat with an intraperitoneal dose of ketamine hydrochloride (90 mg/kg) and xylazine (10 mg/kg). Apply a small amount of ocular lubricant to each eye to prevent corneal dessication. Once the animal&#8217;s respiration is steady and there is no longer any response to a firm toe pinch, the rat has reached a level of anesthesia sufficient for surgery. An acceptable level of anesthesia can be further confirmed by the presence of deep, regular respiration, and normal color of the eye (versus pallor, which might suggest inadequate cardiovascular perfusion).</li>
	<li>Shave the ventral abdomen with a commercial clippers from the pubis to the sternum and from the lateral margins on the ventrum. Remove the clipped hair by applying a moist paper towel to the area.</li>
	<li>Scrub the shaved area with an iodophor antiseptic followed by a wipe with 70% ethanol. Apply surgical drapes so that a window for the incision can be isolated from potential contamination.</li>
	<li>Don a sterile surgical gown, a face mask, and a hair bonnet. Thoroughly wash and scrub hands with soap and don sterile surgical gloves.</li>
</ol>
3. Creation and Repair of the Ventral Abdominal Hernia
<ol>
	<li>Prior to surgery, sterilize surgical instruments by autoclave.</li>
	<li>Using a scalpel, make an approximately 3 - 4 cm vertical incision along the ventral midline down to the level of the abdominal musculature. Lift the abdominal wall gently with a forceps and create a small opening through the linea alba. To protect the underlying viscera, insert a forceps through the hole and into the abdominal cavity to serve as a protective guide as the muscle wall is incised to create an approximately 2 cm x 2 cm full thickness defect, cutting approximately 1 cm lateral from each side of the midline on both ends of the linea alba incision.</li>
	<li>Lay a pre-cut 2 cm x 2 cm section of mechanical bridging material, 4-layer SIS in this case (Figure 1), over the defect. 
	Note: Alternatively, an underlay approach can be used in which the section of material is placed into the abdominal cavity to cover the defect from the interior aspect.</li>
	<li>Suture the four corners of the bridging material to the abdominal wall muscle using 4-0 silk or nylon in a simple interrupted suture pattern. 
	Note: The use of non-absorbable suture allows easy identification of the implanted material upon harvest versus absorbable suture material.</li>
	<li>Secure each edge of the material with three additional sutures spaced equally. Examine the entire circumferential edge to ensure that significant gaps do not exist that might allow passage of viscera through the abdominal wall; and, if such gaps exist, place additional sutures to further secure the margin.</li>
	<li>Close the subcutaneous tissue with 4-0 absorbable suture in a simple interrupted pattern. Close the skin with surgical staples, though suture applied in a simple interrupted pattern may also be used.</li>
	<li>Do not leave the animal unattended until it has regained sufficient consciousness to regain sternal recumbency; and do not return an animal that has undergone surgery to the company of other animals until it has fully recovered from anesthesia. During this time, keep the animal in a warm area to prevent hypothermia.</li>
	<li>Examine animals daily that have undergone this procedure to ensure full recovery and normal healing. Remove skin staples with a staple remover 7 - 10 days following surgery.</li>
</ol>
4. Harvest of the Implanted Material
<ol>
	<li>Consistent with the needs of the study, euthanize, with carbon dioxide overdose administered at a chamber fill rate of 10 to 30%, the animals at a pre-determined time, often 21 - 28 days following surgery.</li>
	<li>Using standard necropsy tools, open the skin and abdominal wall and expose the area of the implant. 
	Note: If non-absorbable sutures were used to secure the implant, the material can be identified by the original sutures (Figure 2).</li>
	<li>Observe any gross pathological observations including any adverse reactions (e.g., wound dehiscence, abscess, seroma, hematoma) and adhesion extent and tenacity to the underlying viscera.</li>
	<li>Remove the implant site by cutting a wide section of tissue that includes the implant. Place the sample in a fixative, such as 10% neutral buffered formalin, in preparation for processing for histological examination and characterization.</li>
</ol>

<ol>
	<li>Poussier, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+available+prosthetic+material+for+abdominal+wall+repair.">A review of available prosthetic material for abdominal wall repair.</a> J. Visc. Surg. 150 (1), 52-59 (2013).</li><li>Hjort, H., Mathisen, T., Alves, A., Clermont, G., Boutrand, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-year+results+from+a+preclinical+implantation+study+of+a+long-term+resorbable+surgical+mesh+with+time-dependent+mechanical+characteristics.">Three-year results from a preclinical implantation study of a long-term resorbable surgical mesh with time-dependent mechanical characteristics.</a> Hernia. 16 (2), 191-197 (2012).</li><li>Ko, R., Kazacos, E. A., Snyder, S., Ernst, D. M. J., Lantz, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+strength+comparison+of+small+intestinal+submucosa+body+wall+repair.">Tensile strength comparison of small intestinal submucosa body wall repair.</a> J. Surg. Res. 135 (1), 9-17 (2006).</li><li>Garcia-Moreno, F., 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=Comparing+the+host+tissue+response+and+peritoneal+behavior+of+composite+meshes+used+for+ventral+hernia+repair.">Comparing the host tissue response and peritoneal behavior of composite meshes used for ventral hernia repair.</a> J. Surg. Res. 193 (1), 470-482 (2015).</li><li>Soiderer, E. E., Lantz, G. C., Kazacos, E. A., Hodde, J. P., Wiegand, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+study+of+three+collagen+materials+for+body+wall+repair.">Morphologic study of three collagen materials for body wall repair.</a> J. Surg. Res. 118 (2), 161-175 (2004).</li><li>Liu, Z., Tang, R., Zhou, Z., Song, Z., Wang, H., Gu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+porcine-derived+materials+for+repairing+abdominal+wall+defect+in+rats.">Comparison of two porcine-derived materials for repairing abdominal wall defect in rats.</a> PLOS One. 6 (5), e20520 (2011).</li><li>Zhang, J., Wang, G. Y., Xiao, Y. P., Fan, L. Y., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biomechanical+behavior+and+host+response+to+porcine-derived+small+intestine+submucosa,+pericardium+and+dermal+matrix+acellular+grafts+in+a+rat+abdominal+defect+model.">The biomechanical behavior and host response to porcine-derived small intestine submucosa, pericardium and dermal matrix acellular grafts in a rat abdominal defect model.</a> Biomaterials. 32 (29), 7086-7095 (2011).</li><li>Ma, J., Sahoo, S., Baker, A. R., Derwin, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+muscle+regeneration+with+a+dermis/small+intestinal+submucosa+scaffold+in+a+rat+full-thickness+abdominal+wall+defect+model.">Investigating muscle regeneration with a dermis/small intestinal submucosa scaffold in a rat full-thickness abdominal wall defect model.</a> J.Biomed.Mater.Res.Part B. 103 (2), 355-364 (2015).</li><li>Greca, F. H., Souza-Filho, Z. A., Giovanni, A., Rubin, M. R., Kuenzer, R. F., Reese, F. B., Araujo, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+porosity+on+the+integration+histology+of+two+polypropylene+meshes+for+the+treatment+of+abdominal+wall+defects+in+dogs.">The influence of porosity on the integration histology of two polypropylene meshes for the treatment of abdominal wall defects in dogs.</a> Hernia. 12 (1), 45-49 (2008).</li><li>Hiles, M., Record-Ritchie, R. D., Altizer, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+biologic+grafts+effective+for+hernia+repair?+A+systematic+review+of+the+literature.">Are biologic grafts effective for hernia repair? A systematic review of the literature.</a> Surg. Innov. 16 (1), 26-37 (2009).</li><li>Helton, W. S., Fisichella, P. M., Berger, R., Horgan, S., Espat, N. J., Abcarian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+outcomes+with+small+intestinal+submucosa+for+ventral+abdominal+hernia.">Short-term outcomes with small intestinal submucosa for ventral abdominal hernia.</a> Arch. Surg. 140 (6), 560-562 (2005).</li><li>Franklin, M. E., Trevino, J. M., Portillo, G., Vela, I., Glass, J. L., Gonzalez, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+porcine+small+intestinal+submucosa+as+a+prosthetic+material+for+laparascopic+hernia+repair+in+infected+and+potentially+contaminated+fields:+long-term+follow-up.">The use of porcine small intestinal submucosa as a prosthetic material for laparascopic hernia repair in infected and potentially contaminated fields: long-term follow-up.</a> Surg. Endosc. 22 (9), 1941-1946 (2008).</li><li>Clarke, K. M., Lantz, G. C., Salisbury, S. K., Badylak, S. F., Hiles, M. C., Voytik, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestine+submucosa+and+polypropylene+mesh+for+abdominal+wall+repair+in+dogs.">Intestine submucosa and polypropylene mesh for abdominal wall repair in dogs.</a> J. Surg. Res. 60 (1), 107-114 (1996).</li><li>Sarikava, A., Record, R., Wu, C. C., Tullius, B., Badylak, S. F., Ladisch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+activity+associated+with+extracellular+matrices.">Antimicrobial activity associated with extracellular matrices.</a> Tissue Eng. 8 (1), 63-71 (2002).</li><li>Serra, R., Grande, R., Butrico, L., Rossi, A., Settimio, U. F., Caroleo, B., Amato, B., Gallelli, L., de Franciscis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+wound+infections:+the+role+of+Pseudomonas+aeruginosa+and+Staphylococcus+aureus.">Chronic wound infections: the role of Pseudomonas aeruginosa and Staphylococcus aureus.</a> Expert Rev. Anti. Infect. Ther. 13 (5), 605-613 (2015).</li></ol>

One of the authors (CJ) is employed by Cook Biotech, Inc., the manufacturer of medical grade SIS.

Materials for the repair of ventral abdominal hernia are of great interest, particularly those that provide an initial mechanical bridge and are then able to incorporate into host tissue. In this regard, a variety of materials have been investigated, including SIS, porcine acellular dermal matrix, and porcine pericardium.6,7 These materials represent the extracellular matrices of those tissues and act as scaffolds into which cells can migrate and proliferate, thus facilitating tissue incorporation.
Evaluation of hernia repair materials requires an animal model that is easily handled and has intact healing ability. The rat ventral hernia model allows for ease of handling in studies to assess repair materials in vivo. It has been demonstrated that this model could be used for comparative histological assessment of host response to implanted hernia repair materials.6 This is particularly essential for biomaterials, such as SIS, that undergo incorporation into the host tissue as part of the repair mechanism.
The rat ventral abdominal hernia repair model can be easily performed in a very reproducible manner. Proper aseptic technique is essential for success, as contaminated surgical fields can alter the healing response and thus complicate interpretation of experimental results.15 The protocol described here is designed to provide an individual experienced in animal surgery with a method to successfully undertake studies requiring hernia creation and repair.
The procedure described here involves an outlay approach in which the repair material is attached to the external edges of the abdominal wall defect. It is also possible to use an inlay approach in which the repair material is attached to the internal aspect of the defect. Hernia repair with mesh via laparoscopy is typically achieved with an inlay approach. The inlay method allows greater direct contact of the implant with abdominal viscera, thus somewhat increasing the possibility of adhesiogenesis between implant and viscera. Because post-surgical adhesion formation is an important clinical issue, studies that wish to closely examine adhesiogenesis may wish to use the inlay approach.
Though rats generally recover very quickly following the surgery, some possibility for post-operative complication exists. For example, some rats will remove skin sutures, even staples. If the incision wound is found to be open, the rat should either be euthanized or re-anesthetized and the skin closed. Though rare, if both the skin and abdominal wall are found to have open gaps, the animal should be euthanized. In all such cases, the attending veterinarian should be promptly informed of such animals.
The model described here has a number of advantages as indicated earlier. However, it should be recognized that modeling of ventral abdominal hernia in a quadraped, such as a rat, limits translation to man. Further, because many clinically relevant hernias are believed to be associated with co-morbidities such as diabetes, obesity, and connective tissue defects, evaluation of repair strategies in a normal animal could complicate the translational value of the model. Nonetheless, the rat model allows relatively rapid and easy evaluation of hernia repair materials.
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. Likewise, the evaluation of induced pluripotent stem cell methods to aid tissue repair will likely find utility in this model.
Critical steps for successful use of this model are centered on care of the animal and surgical skill. Aseptic technique must be used as a way to minimize the chance of infection. Proper anesthesia and analgesia are essential, as well as post-operative monitoring to ensure that animals are comfortable and that the surgical incision remains closed. Importantly, when securing the biomaterial to the edges of the abdominal wall defect, the surgeon must establish that all edges are secure with no openings that might allow passage of abdominal viscera into the subcutaneous space.

Abdominal wall hernia is a commonly encountered clinical issue which may occur as a result of congenital defect, traumatic injury, or failed closure of surgical wounds involving the abdominal wall. Repair commonly involves using an implant to reinforce the abdominal wall, with a reduced rate of recurrence noted in patients treated with an implant compared to those in whom the abdominal wall is simply closed with suture.1
Surgical hernia repair often requires mechanical bridging of the defect with an implanted material. In this regard, both synthetic and natural materials have been used for hernia repair. Many synthetic materials are typically non-absorbable and include polypropylene, polyethylene terephthalate polyester, and expanded polytetrafluoroethylene, while other synthetic implants combine one of the non-absorbable materials with an absorbable polymer such as polygalactine.1 In contrast, natural material implants are generally derived from either animal or human cadaveric sources. Natural materials used for hernia repair are typically rich in collagen and act as a scaffold for ingrowth and integration of host tissue.
The desire to develop and optimize new materials for ventral abdominal hernia repair requires an appropriate animal model for evaluation of candidate materials. Studies have been conducted in a variety of species, including sheep,2 pigs,3 rabbits,4 rats,5,6,7,8 and dogs.9&#160; The advantage of using rats for such studies is that they offer an easily handled model available in a variety of inbred strains to allow for control of genetic variability. In addition, the smaller overall body surface area of the rat compared to the other species listed above permit evaluation of materials that may be experimental in nature and, therefore, not abundant.&#160; Further, reduced costs associated with rats versus other species allow for relatively increased ability to screen candidate materials for use in repair of ventral abdominal hernia.&#160; Considerations for materials typically include ease of handling by the surgeon, strength, ability to incorporate into host tissue, biocompatibility, and resistance to infection.
As an example, porcine small intestinal submucosa (SIS) is a natural biomaterial that has been used for a wide variety of tissue repair indications, including repair of ventral abdominal, inguinal, and diaphragmatic hernias.10,11,12 SIS is rapidly incorporated into host tissue and has been found to be generally resistant to infection and so the material has particular utility in contaminated fields.13,14&#160; In the rat model, SIS has demonstrated ease of handling, good tensile strength as determined by the&#160; uniaxial tensile failure test, and promotion of tissue ingrowth in terms of collagen deposition and neovascularization.6,7
Accurate surgical bridging of the defect is an essential feature of productive modeling when evaluating candidate repair materials. The animal must be sufficiently anesthetized and aseptic technique strictly followed. Further, an abdominal wall defect of standard size must be carefully created and then bridged in a manner that sufficiently secures the material to the margins of the defect.8 This protocol provides a standard method to be followed for surgical creation, and repair of, a ventral abdominal hernia using SIS as an example bridging material in a rat.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Isoflurane (Isoflo)</td>
			<td style="width:71px;">Henry Schine Animal Health</td>
			<td align="right" style="width:119px;">17579</td>
			<td style="width:167px;">gas anesthetic</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Optixcare Eye Lubricant</td>
			<td style="width:71px;">Henry Schine Animal Health</td>
			<td style="width:119px;">OPX4240</td>
			<td>Ocular lubricant</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Oster clippers</td>
			<td style="width:71px;">Henry Schine Animal Health</td>
			<td align="right" style="width:119px;">6092</td>
			<td>hair clippers</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Betadine surgical scrub</td>
			<td style="width:71px;">Henry Schine Animal Health</td>
			<td align="right" style="width:119px;">1618</td>
			<td>antiseptic iodophor</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Sterile scalpel</td>
			<td style="width:71px;">Henry Schine Animal Health</td>
			<td align="right" style="width:119px;">329</td>
			<td>#10 scalpel</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">9&#34; x 12&#34; disposable surgical drape</td>
			<td style="width:71px;">Braintree Scientific, Inc.</td>
			<td style="width:119px;">SP-RPS</td>
			<td>Surgical drape</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">4-0 nylon suture</td>
			<td style="width:71px;">Braintree Scientific, Inc.</td>
			<td style="width:119px;">SUT 812</td>
			<td>Suture material</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">4-0 Absorbable Suture</td>
			<td style="width:71px;">Henry Schine Animal Health</td>
			<td align="right" style="width:119px;">29242</td>
			<td>Synthetic absorbable suture</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">9 mm Autoclips and applier</td>
			<td style="width:71px;">Braintree Scientific, Inc.</td>
			<td style="width:119px;">ACS KIT</td>
			<td>Surgical staples for closure of skin incision</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Torbugesic/analgesic</td>
			<td style="width:71px;">Henry Schine Animal Health</td>
			<td align="right" style="width:119px;">12084</td>
			<td>Butorphanol tartarate for post-operative analgesia</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Small intestinal submucosa (SIS)</td>
			<td style="width:71px;">Cook Biotech, Inc.</td>
			<td style="width:119px;"></td>
			<td>Implant material for bridging experimental abdominal wall defect</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rats</td>
			<td style="width:71px;">Harlan, Inc.</td>
			<td style="width:119px;">Sprague Dawley</td>
			<td>Animal for hernia modeling</td>
		</tr>
	</tbody>
</table>

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.

By 28 days after implantation, SIS typically demonstrates good incorporation into host tissue (Figure 2). In most cases, there is residual SIS apparent, though the tissue must often be explored to identify the implanted material. Histological analyses confirmed the gross pathological observations, illustrating good tissue incorporation with primarily fibrovascular tissue and very little residual SIS noted (Figure 3). These results demonstrate the utility of the rat ventral abdominal hernia model. The rat is easily obtained and maintained, and generally recovers quickly following surgery. The tissue incorporation into the implanted biomaterial indicates biocompatibility and demonstrates the ability of the SIS to serve as a lattice for tissue ingrowth and repair. Even with substantial tissue incorporation, the site of the implant can be readily identified by the sutures used to secure the material to the abdominal wall. As a result, hernia repair materials can be evaluated in a consistent and relatively expeditious manner.
<img alt="Figure 1" src="/files/ftp_upload/53587/53587fig1.jpg" /> 
Figure 1: 2 x 2 cm Section of SIS Used for Repair of Experimental Ventral Abdominal Hernia. The SIS material is an acellular extracellular matrix derived from porcine small intestinal submucosa. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53587/53587fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53587/53587fig2.jpg" /> 
Figure 2: Incorporated SIS 28 Days After Implantation to Bridge a Ventral Abdominal Hernia in a Rat. Following euthanasia, the ventral abdominal skin was reflected to expose the implanted material that is outlined by the silk sutures used to secure the SIS. Note the ingrowth of tissue as the SIS implant has been incorporated into host tissue. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53587/53587fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53587/53587fig3.jpg" /> 
Figure 3: Photomicrograph of Tissue 28 Days Following Implantation of SIS to Bridge an Experimental Ventral Abdominal Hernia. The SIS has largely been incorporated into the host tissue. The arrows show a very small strand of residual SIS in the section (H &#38; E, 40X). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53587/53587fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Use of a Rat Model to Study Ventral Abdominal Hernia Repair at JoVE.com

Use of a Rat Model to Study Ventral Abdominal Hernia Repair

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 ...

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9.1K Views.  University of Minnesota. 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.

Video: Use of a Rat Model to Study Ventral Abdominal Hernia Repair

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two tissue edges1,2. These are then irradiated by a laser that is selectively absorbed by the RB. The resulting photochemical reactions supposedly crosslink the collagen fibers in the tissue with minimal heat production3. In this report, RB has been incorporated in thin chitosan films to fabricate a novel tissue adhesive that is laser-activated. Adhesive films, based on chitosan and containing ~0.1 wt% RB, are fabricated and bonded to calf intestine and rat tibial nerves by a solid state laser (&lambda;=532 nm, Fluence~110 J/cm2, spot size~0.5 cm). A single-column tensiometer, interfaced with a personal computer, is used to test the bonding strength. The RB-chitosan adhesive bonds firmly to the intestine with a strength of 15 &plusmn; 6 kPa, (n=30). The adhesion strength drops to 2 &plusmn; 2 kPa (n=30) when the laser is not applied to the adhesive. The anastomosis of tibial nerves can be also completed without the use of sutures. A novel chitosan adhesive has been fabricated that bonds photochemically to tissue and does not require sutures.

Sutures are usually needed to repair tissue during surgical procedures. However, their application can be problematic as they are invasive and may damage tissue. The fabrication and application methods of a novel tissue adhesive are here reported. This adhesive film is laser-activated and does not require the use of sutures.

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two ...

Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This technique allows for easier venous cuff-anatomoses after a reasonable learning curve. The device is composed of two handles, which are carved out from scalpel blades, one approximator, which is obtained by modifying Kocher's forceps, and cuffs designed from fine-bore polyethylene tubing. The whole process can be performed at a low-cost using common laboratory material. The present report provides a step-by-step protocol for the design of the required pieces and includes stencils.

We describe the design of the &#x201C;quick-linker&#x201D; device for easier orthotopic rat liver transplantation.

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This ...

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 ...

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 ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

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) ...