This research was supported by the Guangdong Science and Technology Plan Project (grant number: 2021A1515410004) and the National Key Clinical Discipline (grant number: [2012]649).

The protocol was carried out in accordance with the tenets of the Declaration of Helsinki and approved by the Ethics Review Committee of the Sixth Affiliated Hospital of Sun Yat-sen University.
The patients and families were informed of the purpose of shooting and making the surgical video, and informed consent was obtained.
1. Patient data and grouping
NOTE: From January 2018 to June 2020, laparoscopic incisional hernia repair using the IPOM method was performed during gastrointestinal, hernia, and abdominal surgery at the Sixth Affiliated Hospital of Sun Yat-sen University. After obtaining informed consent, a total of 84 patients with incisional hernias were included in the study.
<ol>
	<li>Enroll patients based on the inclusion criteria (adult patients diagnosed with incisional hernias) and exclusion criteria (age &#8804;18 years or &#8805;80 years; emergency operation; strangulated incisional hernia; recurrent incisional hernia; the presence of severe organ dysfunction). 
	NOTE: The characteristics of the two groups, including the patient&#39;s age, sex, and body mass index (BMI), the length of time the hernia was present, and the maximum size of the hernia ring defect, are shown in Table 1. There were no significant differences in characteristics between the two groups (all P &#62; 0.05).</li>
	<li>All the operations were performed by the same group of surgeons, who received standardized training and had rich experience in laparoscopic surgery.</li>
</ol>
2. Necessary preparation and examination before the operation
<ol>
	<li>Carry out preoperative examinations, including routine blood tests, blood biochemistry tests, routine urine tests, a routine stool test, a chest X-ray, an electrocardiogram, and an abdominal CT.</li>
	<li>For a giant incisional hernia, use preoperative botulinum toxin A (BTA) and preoperative progressive pneumoperitoneum (PPP) for the surgical preparation11.</li>
	<li>Administer fentanyl at 4 &#181;g/kg using another pump at a rate of 250 &#181;g/min simultaneously with propofol administration. Intravenously inject cisatracurium (0.2 mg/kg) after the patient is anesthetized.
	<ol>
		<li>Subsequently, intubate the patient with the help of an experienced anesthesiologist 4 min after the muscle relaxant injection. Next, mechanically ventilate the patient with sevoflurane 1% inhalation with the following respiratory parameters: tidal volume, 8 mL/kg; respiratory rate, 12 breaths per minute. 
		NOTE: The anesthetic maintenance drugs were sevoflurane 1%-3%, propofol 1-3 mg/kg/h, remifentanil 0.05-0.3 &#181;g/kg/min, cisatracurium 0.15-0.2 mg/kg, and 1/5-1/3 of the additional induction amount every 0.5-1 h12.</li>
	</ol>
	</li>
	<li>Use 0.5% PVP-I disinfectant to disinfect the surgical area. The upper part of the disinfection range should reach the nipple line on both sides, the lower part should reach the pubic symphysis, and both sides should reach the midaxillary line.</li>
	<li>After endotracheal intubation, place the trocars using the method of &#65279;modelized trocar arrangement. Determine the locations of the trocar placement based on the preoperative computed tomography (CT) images and the assessment of the abdominal adhesions13. 
	NOTE: For example, the case in this video is a lower abdominal incision hernia, which requires a total of five puncture tubes. A 12 mm puncture device is placed 10 cm above the belly button, 12 mm and 5 mm puncture tubes are placed at the left and right clavicular midlines, and 5 mm puncture tubes are placed at the left and right axillary midlines.</li>
	<li>Establish carbon dioxide pneumoperitoneum, and maintain the pneumoperitoneum pressure at 13 mmHg.</li>
	<li>Explore the whole abdominal cavity, evaluate the degree of abdominal adhesion around the hernia ring, and separate the adhesion.</li>
	<li>Close the defect of the hernia ring by continuous suture with a 1-0 barbed suture.</li>
</ol>
3. Measurement of the size of the hernia ring defect and anti-adhesion mesh marking
<ol>
	<li>Select an appropriate size anti-adhesion mesh (see Table of Materials) according to the size of the hernia ring defect, and then mark the mesh with a sterile marking pen as described below. 
	NOTE: A commercially available anti-adhesion mesh was used here, which contains a polypropylene mesh on the anterior side with an absorbable hydrogel barrier on the posterior side for laparoscopic hernia repair. The hydrogel barrier on the mesh prevents adhesion, and the mesh on this side should face the viscera.
	<ol>
		<li>Measure and mark the approximate range of the incisional hernia on the abdominal wall surface with a sterile ruler and marking pen (Figure 1A).The size of the hernia ring defect can also be measured by a preoperative CT examination.</li>
		<li>Place the ruler parallel to the longitudinal axis of the hernia defect, and measure the maximum longitudinal length of the defect (Figure 1B).</li>
		<li>Select an appropriately sized anti-adhesion mesh according to the size of the hernia ring defect. Ensure that the coverage of the mesh exceeds the edge of the defect by at least 5 cm. For example, for an incisional hernia defect of size 7 cm x 5 cm, use a mesh approximately 20 cm x 15 cm in size (Figure 1C).</li>
		<li>Mark the longitudinal length of the defect on the mesh, and mark the nail gun fixation points at intervals of 5 cm on the longitudinal axis. Then, extend the fixation points more than 5 cm along the marked line to the edge of the mesh, which pertains to the &#34;alignment&#34; (Figure 1D).</li>
		<li>Mark the nail gun fixation points evenly every 2-3 cm along the edge of the mesh, which pertains to the &#34;contraposition&#34; (Figure 1E).</li>
		<li>Finally, ensure that the fixing points of the nail gun are uniformly marked 2 cm from the longitudinal axis of the defect on both sides, with an interval of 3 cm (Figure 1F).</li>
	</ol>
	</li>
</ol>
4. Mesh placement method
<ol>
	<li>Roll the mesh such that the anti-adhesion surface will face the abdominal wall. Place the rolled mesh into the abdominal cavity through the 12 mm puncture hole, and then unfold the mesh under laparoscopic guidance (see Table of Materials, (Figure 2B).</li>
	<li>Reduce the pneumoperitoneum pressure to 8-10 mmHg.</li>
	<li>Ensure that the marked line of the unrolled mesh overlaps the longitudinal axis of the hernia ring defect (Figure 2C).</li>
	<li>Fix the marked points on the longitudinal axis of the mesh to the abdominal wall with non-absorbable nails using a nail gun (see Table of Materials; (Figure 2D).</li>
	<li>Fix the edge of the mesh to the abdominal wall along the marked points on the edge of the mesh with non-absorbable nails using a nail gun (Figure 2E).</li>
	<li>Fix the mesh on the abdominal wall along the marked points on both sides of the longitudinal axis of the mesh with absorbable nails using a nail gun (Figure 2F).</li>
	<li>For the control group, flatten the mesh to cover the abdominal wall defect and fix the mesh using the double-loop mesh fixation method9,14.</li>
	<li>For the double-ring fixing method, do not mark the mesh.
	<ol>
		<li>First, place the fixing nails along the edge of the mesh to ensure that the distance between the nails and the edge of the mesh is about 2-4 mm and that the distance between the nails is about 2-3 cm.</li>
		<li>Then, fix the mesh along the outer edge of the hernia ring defect about 2 cm away from it, with a distance between the nails of 3-5 cm 
		&#8203;NOTE: See Figure 3A-D for this method.</li>
	</ol>
	</li>
</ol>
5. Follow-up
<ol>
	<li>Perform postoperative follow-up, including outpatient visits and telephone consultations, for 3 months to 24 months. 
	NOTE: In this study, the median follow-up time was 12 months. A physical examination and abdominal wall color ultrasound were performed in the first month after the operation, and an abdominal CT was performed at 3 months, 12 months, and 24 months after the operation.The follow-up time was 24 months after the operation.After all the follow-ups, the case data were collected and compared.</li>
	<li>Record the occurrence of seromas, hernia recurrence, chronic pain, and mesh infection. 
	Seroma can be diagnosed by an abdominal wall color ultrasound, hernia recurrence and mesh infection by an abdominal CT, and chronic pain by a pain rating scale.In particular, carry out an abdominal CT examination with patients 3 months after surgery, and then compare the results with the preoperative abdominal CT images to evaluate the surgical treatment effects and whether there is a recurrence of the incisional hernia (Figure 4A-F).</li>
</ol>
6. Statistical analysis
<ol>
	<li>Compare the time required for mesh placement, seroma formation, mesh infection, hernia recurrence, chronic pain, the length of hospital stay, and the hospital costs between the two groups. 
	NOTE: In this study, the measurement data (age, BMI, disease time, mesh placement time, length of hospital stay, and hospitalization costs) were expressed as mean &#177; standard deviation, and the count data (gender, maximum defect of the hernia ring, seroma, mesh infection, hernia recurrence, and chronic pain) were expressed as a count and percentage.</li>
	<li>Compare the measurement data between the groups using a t-test, and compare the count data using a chi-square test. A value of P &#60; 0.05 was considered to indicate a statistically significant difference in this study.</li>
</ol>

<ol>
	<li>Misiakos, E., Patapis, P., Zavras, N., Tzanetis, P., Machairas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+trends+in+laparoscopic+ventral+hernia+repair.">Current trends in laparoscopic ventral hernia repair.</a> Journal of the Society of Laparoscopic &amp; Robotic Surgeons. 19 (3), 2015 (2015).</li><li>Raakow, 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=A+comparison+of+laparoscopic+and+open+repair+of+subxiphoid+incisional+hernias.">A comparison of laparoscopic and open repair of subxiphoid incisional hernias.</a> Hernia. 22 (6), 1083-1088 (2018).</li><li>Warren, J., Love, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incisional+hernia+repair:+Minimally+invasive+approaches.">Incisional hernia repair: Minimally invasive approaches.</a> The Surgical Clinics of North America. 98 (3), 537-559 (2018).</li><li>Judy, J., Michael, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+versus+open+ventral+hernia+repair.">Laparoscopic versus open ventral hernia repair.</a> The Surgical Clinics of North America. 88 (5), 1083-1100 (2008).</li><li>Muysoms, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IPOM:+History+of+an+acronym.">IPOM: History of an acronym.</a> Hernia. 22 (5), 743-746 (2018).</li><li>Tim, T., 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=Prospective+analysis+of+ventral+hernia+repair+using+the+Ventralight&#8482;+ST+hernia+patch.">Prospective analysis of ventral hernia repair using the Ventralight&#8482; ST hernia patch.</a> Surgical Technology International. 23, 113-116 (2013).</li><li>Henriksen, N., 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=Open+versus+laparoscopic+incisional+hernia+repair:+Nationwide+database+study.">Open versus laparoscopic incisional hernia repair: Nationwide database study.</a> BJS Open. 5 (1), (2021).</li><li>K&#246;ckerling, F., Schug-Pass, C., Bittner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+word+of+caution:+Never+use+tacks+for+mesh+fixation+to+the+diaphragm.">A word of caution: Never use tacks for mesh fixation to the diaphragm.</a> Surgical Endoscopy. 32 (7), 3295-3302 (2018).</li><li>Olmi, S., 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=Prospective+clinical+study+of+laparoscopic+treatment+of+incisional+and+ventral+hernia+using+a+composite+mesh:+Indications,+complications+and+results.">Prospective clinical study of laparoscopic treatment of incisional and ventral hernia using a composite mesh: Indications, complications and results.</a> Hernia. 10 (3), 243-247 (2006).</li><li>Ning, 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=Application+of+&amp;#34;contraposition+and+alignment&amp;#34;+mesh+fixation+in+laparoscopic+incisional+hernia+repair.">Application of &amp;#34;contraposition and alignment&amp;#34; mesh fixation in laparoscopic incisional hernia repair.</a> Chinese Journal of General Surgery. 31 (4), 474-480 (2022).</li><li>Fu-Xin, T., 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=Botulinum+toxin+A+facilitated+laparoscopic+repair+of+complex+ventral+hernia.">Botulinum toxin A facilitated laparoscopic repair of complex ventral hernia.</a> Frontiers in Surgery. 8, 803023 (2022).</li><li>Lihong, 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=Observer's+assessment+of+alertness/sedation-based+titration+reduces+propofol+consumption+and+incidence+of+hypotension+during+general+anesthesia+induction:+A+randomized+controlled+trial.">Observer's assessment of alertness/sedation-based titration reduces propofol consumption and incidence of hypotension during general anesthesia induction: A randomized controlled trial.</a> Science Progress. 104 (4), 368504211052354 (2021).</li><li>Zhou, 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=Application+of+modelized+port+arrangement+based+on+data+analysis+and+calculation+in+laparoscopic+repair+of+abdominal+wall+incisional+hernia.">Application of modelized port arrangement based on data analysis and calculation in laparoscopic repair of abdominal wall incisional hernia.</a> Chinese Journal of General Surgery. 31 (4), 449-456 (2022).</li><li>Bittner, R., 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=Update+of+Guidelines+for+laparoscopic+treatment+of+ventral+and+incisional+abdominal+wall+hernias+(International+Endohernia+Society+(IEHS))+-+Part+A.">Update of Guidelines for laparoscopic treatment of ventral and incisional abdominal wall hernias (International Endohernia Society (IEHS)) - Part A.</a> Surgical Endoscopy. 33 (10), 3069-3139 (2019).</li><li>Petersen, S., 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=Deep+prosthesis+infection+in+incisional+hernia+repair:+Predictive+factors+and+clinical+outcome.">Deep prosthesis infection in incisional hernia repair: Predictive factors and clinical outcome.</a> The European Journal of Surgery. 167 (6), 453-457 (2001).</li><li>Montgomery, A., 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=Evidence+for+replacement+of+an+infected+synthetic+by+a+biological+mesh+in+abdominal+wall+hernia+repair.">Evidence for replacement of an infected synthetic by a biological mesh in abdominal wall hernia repair.</a> Frontiers in Surgery. 2, 67 (2015).</li><li>Mathes, T., 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=Mesh+fixation+techniques+in+primary+ventral+or+incisional+hernia+repair.">Mesh fixation techniques in primary ventral or incisional hernia repair.</a> The Cochrane Database of Systematic Reviews. 5 (5), (2021).</li><li>Rieder, E., 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=Mesh+fixation+in+laparoscopic+incisional+hernia+repair:+Glue+fixation+provides+attachment+strength+similar+to+absorbable+tacks+but+differs+substantially+in+different+meshes.">Mesh fixation in laparoscopic incisional hernia repair: Glue fixation provides attachment strength similar to absorbable tacks but differs substantially in different meshes.</a> Journal of the American College of Surgeons. 212 (1), 80-86 (2011).</li><li>Stoikes, N., 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=Biomechanical+evaluation+of+fixation+properties+of+fibrin+glue+for+ventral+incisional+hernia+repair.">Biomechanical evaluation of fixation properties of fibrin glue for ventral incisional hernia repair.</a> Hernia. 19 (1), 161-166 (2015).</li><li>Bansal, V., 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+prospective+randomized+study+comparing+suture+mesh+fixation+versus+tacker+mesh+fixation+for+laparoscopic+repair+of+incisional+and+ventral+hernias.">A prospective randomized study comparing suture mesh fixation versus tacker mesh fixation for laparoscopic repair of incisional and ventral hernias.</a> Surgical Endoscopy. 25 (5), 1431-1438 (2011).</li><li>Eriksen, 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=Pain,+quality+of+life+and+recovery+after+laparoscopic+ventral+hernia+repair.">Pain, quality of life and recovery after laparoscopic ventral hernia repair.</a> Hernia. 13 (1), 13-21 (2009).</li><li>Bageacu, S., 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=Laparoscopic+repair+of+incisional+hernia:+A+retrospective+study+of+159+patients.">Laparoscopic repair of incisional hernia: A retrospective study of 159 patients.</a> Surgical Endoscopy. 16 (2), 345-348 (2002).</li><li>Taylor, 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=Laparoscopic+inguinal+hernia+repair+without+mesh+fixation,+early+results+of+a+large+randomised+clinical+trial.">Laparoscopic inguinal hernia repair without mesh fixation, early results of a large randomised clinical trial.</a> Surgical Endoscopy. 22 (3), 757-762 (2008).</li><li>Baker, 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=Decreased+re-operation+rate+for+recurrence+after+defect+closure+in+laparoscopic+ventral+hernia+repair+with+a+permanent+tack+fixated+mesh:+A+nationwide+cohort+study.">Decreased re-operation rate for recurrence after defect closure in laparoscopic ventral hernia repair with a permanent tack fixated mesh: A nationwide cohort study.</a> Hernia. 22 (4), 577-584 (2018).</li><li>Beldi, G., 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=Mesh+shrinkage+and+pain+in+laparoscopic+ventral+hernia+repair:+A+randomized+clinical+trial+comparing+suture+versus+tack+mesh+fixation.">Mesh shrinkage and pain in laparoscopic ventral hernia repair: A randomized clinical trial comparing suture versus tack mesh fixation.</a> Surgical Endoscopy. 25 (3), 749-755 (2011).</li><li>Muysoms, 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=Randomized+clinical+trial+of+mesh+fixation+with+&amp;#34;double+crown&amp;#34;+versus+&amp;#34;sutures+and+tackers&amp;#34;+in+laparoscopic+ventral+hernia+repair.">Randomized clinical trial of mesh fixation with &amp;#34;double crown&amp;#34; versus &amp;#34;sutures and tackers&amp;#34; in laparoscopic ventral hernia repair.</a> Hernia. 17 (5), 603-612 (2013).</li><li>Eriksen, 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=Fibrin+sealant+for+mesh+fixation+in+laparoscopic+umbilical+hernia+repair:+1-year+results+of+a+randomized+controlled+double-blinded+study.">Fibrin sealant for mesh fixation in laparoscopic umbilical hernia repair: 1-year results of a randomized controlled double-blinded study.</a> Hernia. 17 (4), 511-514 (2013).</li><li>Erwin, R., 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=Mesh+fixation+in+laparoscopic+incisional+hernia+repair:+Glue+fixation+provides+attachment+strength+similar+to+absorbable+tacks+but+differs+substantially+in+different+meshes.">Mesh fixation in laparoscopic incisional hernia repair: Glue fixation provides attachment strength similar to absorbable tacks but differs substantially in different meshes.</a> Journal of the American College of Surgeons. 212 (1), 80-86 (2011).</li><li>Carbonell, A., 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=Local+injection+for+the+treatment+of+suture+site+pain+after+laparoscopic+ventral+hernia+repair.">Local injection for the treatment of suture site pain after laparoscopic ventral hernia repair.</a> The American Surgeon. 69 (8), 688-692 (2003).</li></ol>

The authors have no conflicts of interest to disclose.

Laparoscopic incisional hernia repair is primarily performed using the IPOM method5, for which the placement and fixation of the mesh are key to achieving good outcomes. If the placement and fixation of the mesh are improper, the mesh will not adhere tightly to the abdominal wall and may be wrinkled or displaced. Improper mesh fixation is associated with seroma formation, abdominal infection, chronic pain, and hernia recurrence. Specifically, the treatment for mesh infections involves puncture and drainage. Postoperative pain associated with mesh infection is often treated conservatively, and poor treatment may necessitate further surgery to remove part or all of the mesh15,16. In order to avoid these complications, it is crucial to master the correct mesh placement and fixation technique. The mesh fixation methods commonly used in laparoscopic incisional hernia repair include hernia nail fixation, suture fixation, and medical adhesive fixation17,18,19. Nail fixation has the advantage of being a simple procedure with a short operation time, and this is the most commonly used method of mesh fixation20. However, the use of hernia nails, especially non-absorbable nails, and the number of nails placed are closely related to the risk of postoperative pain21,22. The use of hernia screws to fix the mesh in certain areas, such as near the external iliac artery and vein in the inguinal region or in the pericardial area of the diaphragm, is associated with an increased risk of massive bleeding23,24. Suture fixation methods include crochet puncture suture fixation, simple suture fixation, and other improved methods25. The main advantages of suture fixation with full abdominal wall suspension are that, with this technique, it is easy to flatten the mesh, and the cost is much lower than that of nail gun fixation. However, using non-absorbable sutures placed through the fascia to fix the mesh is associated with a higher incidence of postoperative pain than hernia nail fixation26. With crochet puncture suture fixation, the crochet needle needs to penetrate the skin into the abdominal cavity, which significantly increases the risk of mesh infection. The use of medical adhesive for mesh fixation is simpler and less expensive, but the fixation with medical adhesive alone is not strong enough, which increases the risk of hernia recurrence27. In addition, the glue block formed during the application of the chemical medical adhesive can affect the process of cell growth and peritoneal mesh formation28. As such, the use of medical adhesive for fixation is only recommended in areas for which the use of nails or sutures may increase the risk of bleeding or pain, such as near blood vessels.
Currently, the most commonly used mesh fixation method is double-loop mesh fixation with hernia nails9,14. However, there are shortcomings to this method. First, it is difficult to accurately locate the spatial axial position of the mesh when placing the mesh, especially for inexperienced surgeons. The mesh center can shift and not completely cover the defect area, which increases the risk of hernia recurrence. Second, the number of hernia nails needed cannot be accurately calculated when placing the mesh, and nail guns typically contain 15 or 30 nails. If the estimated number of hernia nails required is not accurate, some nails may be wasted, thus increasing the medical costs. Third, after the mesh is placed, the blood vessels and nerves cannot be accurately located, which may lead to blood vessel or nerve injury when nailing the mesh.
In view of the shortcomings of the double-loop fixation method, the &#34;contraposition and alignment&#34; mesh fixation method was designed in this study. The results of this study showed that the incidence of seroma and mesh infection, the hernia recurrence rate, the length of postoperative hospital stay, and the hospital costs were similar between the &#34;contraposition and alignment&#34; mesh fixation method and the double-loop fixation method. However, the &#34;contraposition and alignment&#34; mesh fixation method simplifies the surgical procedure by allowing for the mesh to be designed, positioned, and marked in advance, which only takes 1-2 min. After being placed into the abdominal cavity, the mesh is fixed according to the pre-marked positions, which can simplify the placement and reduce the operation time. In addition, marking the areas that overlay vessels and nerves in advance and using absorbable sutures for fixation in these areas can reduce the risk of vascular and nerve injury. It has been reported that in special areas such as the costal margin, the use of a nail gun to fix the mesh or the use of a crochet needle for a full thickness abdominal wall suture can lead to intercostal nerve compression and local muscle ischemia, which may cause chronic pain29. Through a retrospective study, it was found that the &#34;contraposition and alignment&#34; mesh fixation method can significantly shorten the placement time of the mesh. Simultaneously, through the postoperative follow-up, this method was found to reduce the incidence of postoperative chronic pain.
Nevertheless, the &#34;contraposition and alignment&#34; mesh fixation method suffers from a few limitations. This method can only be used with medium or heavyweight anti-adhesion mesh. Lightweight and large anti-adhesion meshes, especially those that are transparent or translucent, are not easy to mark, and it may not be easy to see the marks when placing the mesh. It is also unclear if marking the mesh with a sterile marking pen damages the anti-adhesion coating and, thus, affects mesh adhesion. Additional studies are required to address these issues. Marking in advance when designing and manufacturing the anti-adhesion mesh may help overcome the potential issues arising from using a pen.
There are several key points that should be taken into account when using the &#34;contraposition and alignment&#34; mesh fixation method. When placing the mesh, the length and position of the mesh centerline and the longitudinal axis of the incisional hernia defect should coincide. The longitudinal axis of the defect is the position where the abdominal wall tension is greatest after the hernia ring is closed. The best abdominal wall repair effect can be achieved by fixing the midline based on fixation points that have been designed and marked in advance. Second, the mesh should be fixed according to the nail points marked in advance. When placing the nails, the nail gun, the mesh, and the abdominal wall should be aligned in the same direction to achieve the best fixation effect. Finally, nail gun fixation should not be used in areas containing blood vessels or nerves, and suture fixation can be used to replace nails when necessary to reduce the occurrence of postoperative chronic pain. The ideal effect of the operation is achieved when the mesh is fixed firmly and the use of hernia nails, especially non-absorbable nails, is reduced as much as possible. The mesh edge needs to be fixed with high-strength non-absorbable nails, while absorbable nails can be used on both sides of the longitudinal axis of the mesh to ensure good fixation and reduce postoperative complications.
To achieve the best effect of the mesh placement, the mesh should be completely flattened, the edges should not be curled, and the fixation should be firm such that displacement will not occur. When these conditions are met, the mesh integrates into the peritoneum and abdominal wall tissue rapidly. During the operation, due to the influence of various factors, it is difficult to achieve a rational mesh fixation effect, which requires certain surgical skills. This study shows that the &#34;contraposition and alignment&#34; mesh fixation method offers the advantage of reliable mesh fixation, the technique is easy to learn and master, and the method is associated with a lower risk of chronic pain. Further studies with larger numbers of patients are necessary to confirm the results of this study.

Incisional hernia is a common complication after abdominal surgery and can be properly treated only with surgery1. Compared with traditional open incision herniorrhaphy, laparoscopic herniorrhaphy has the advantages of less surgical trauma, a lower infection rate, and faster postoperative recovery2,3. Currently, laparoscopic herniorrhaphy is the method of choice for the treatment of incisional hernia if there are no contraindications4.
However, laparoscopic herniorrhaphy is technically complex. Intraperitoneal onlay mesh (IPOM) is commonly used in laparoscopic incisional hernia repair, and this involves placing a mesh into the abdominal cavity laparoscopically to cover the hernia defect5. The mesh is a new type of medium-weight monofilament polypropylene mesh covered with a hydrogel barrier on the visceral side6. For laparoscopic incisional hernia repair using the IPOM method, it is necessary to master the placement of the trocars, the techniques to separate intra-abdominal adhesions, the techniques for suturing the incisional hernia, and the methods for placing and fixing the mesh in the abdominal cavity. In particular, if the mesh is not properly placed and fixed, this can result in the recurrence of the hernia, as well as potentially serious complications such as intestinal obstruction and mesh infection7,8. Therefore, mastering the correct mesh fixation technique is an important criterion for achieving a good surgical outcome. 
The traditional method of mesh fixation for incisional hernia is to fix the mesh with a double-ring hernia nail. After the mesh is placed into the abdominal cavity, the edge of the mesh is fixed with a nail gun first, and then the edge of the hernia ring is fixed9. However, this method has poor spatial positioning, and the mesh is prone to displacement, leading to hernia recurrence. By reviewing and analyzing the various mesh fixation methods, a new "contraposition and alignment" method for mesh fixation is proposed and presented in this protocol10. In this method, the size and scope of the incisional hernia are measured in advance, after which the mesh fixation points can be marked in advance. When the mesh is placed into the abdominal cavity during the operation, nail gun fixation and suture fixation can be performed according to the previously marked locations. This method can reduce the difficulty of the operation, the operation time, the medical cost, and the occurrence of complications. In this study, this new method is compared with the conventionally used double-loop hernia nail fixation method for mesh fixation during laparoscopic incisional hernia repair surgery.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-0 Stratafix Symmetric PDS Plus Violet 45cm PS-1&#160;</td>
			<td>ETHICON</td>
			<td>sxpp1a401</td>
			<td>STRATAFIX Symmetric PDS Plus</td>
		</tr>
		<tr>
			<td>3-0 VICRYL suture</td>
			<td>ETHICON</td>
			<td>VCP316</td>
			<td>absorbable suture</td>
		</tr>
		<tr>
			<td>AbsorbaTack Fixation</td>
			<td>Covidien llc</td>
			<td>ABSTACK15</td>
			<td>absorbable nail gun</td>
		</tr>
		<tr>
			<td>Laparoscopic needle holder</td>
			<td>KARL-STORZ</td>
			<td>26173KL</td>
			<td>needle holder</td>
		</tr>
		<tr>
			<td>Laparoscopic separating forceps</td>
			<td>KARL-STORZ</td>
			<td>38651ON</td>
			<td>separating forceps</td>
		</tr>
		<tr>
			<td>Laparoscopic system (OTV-S400)</td>
			<td>Olympus</td>
			<td>CLV-S400_WA4KL530</td>
			<td>4K HD image large screen surgical laparoscope</td>
		</tr>
		<tr>
			<td>ProTack Fixation</td>
			<td>Covidien llc</td>
			<td>174005</td>
			<td>Non absorbable nail gun</td>
		</tr>
		<tr>
			<td>VENTRALIGHT ST</td>
			<td>BARD</td>
			<td>5954810</td>
			<td>Biological anti-adhesion mesh</td>
		</tr>
	</tbody>
</table>

application of a new mesh fixation method in laparoscopic incisional hernia repair

Laparoscopic incisional hernia repair using intraperitoneal onlay mesh (IPOM) is one of the most widely used minimally invasive methods for repairing incisional hernias. The laparoscopic IPOM involves implanting the mesh into the abdominal cavity through laparoscopy to repair an abdominal wall hernia. In the IPOM surgery, after the closure of the hernia ring, an anti-adhesion mesh is placed laparoscopically. The correct placement of this mesh is critical to the success of the method, and surgical skills are required to achieve perfect placement. If the mesh placement is not mastered properly, the operation and anesthesia time will be prolonged. In addition, improper placement of the mesh can lead to serious consequences, such as intestinal obstruction and mesh infection. A "contraposition and alignment" mesh fixation method is described in this study, which involves pre-marking the fixation position of the mesh to reduce the difficulty of mesh placement. A properly placed mesh is completely flat on the peritoneum, the edges are not curled or wrapped, and the mesh is adhered firmly such that there is no displacement after removing the pneumoperitoneum pressure. The "contraposition and alignment" mesh fixation technique offers the advantages of reliable placement of the mesh and fewer complications than other techniques, and it is easy to learn and master. It also allows for positioning the nail gun in advance based on the anatomy of the incisional hernia. This enables the use of the minimum number of nails possible while still ensuring good fixation, which can reduce the occurrence of complications and reduce the cost of surgery. Thus, the mesh fixation method described here is highly suitable for clinical applications based on the aforementioned advantages.

Either &#34;contraposition and alignment&#34; mesh fixation (experimental group) or traditional double-loop hernia nail fixation (control group) was performed for the patients in the study, with 42 patients in each group.
During the hernia repair surgery performed in this study, the anti-adhesion mesh was placed after the hernia ring was sutured. In the experimental group, the &#34;contraposition and alignment&#34; method was used to place the mesh, while the traditional double-loop fixation method was used to place the mesh in the control group.
Section 3 of the protocol details the specific mesh placement methods (Figure 1, Figure 2, and Figure 3). The mesh placement time, the frequency of seroma formation, mesh infection, hernia recurrence, chronic pain, the length of hospital stay, and the hospitalization costs were compared between the experimental and control groups. Figure 4 shows the comparison images before and after the operation. The mean mesh placement time of the experimental group was 32.5 min &#177; 11.6 min and of the control group was 44.7 min &#177; 12.5 min (P &#60; 0.05), indicating a reduction in the time required for mesh placement with the &#34;contraposition and alignment&#34; method used in this study. There were only two cases of chronic pain in the experimental group, which was a significantly lower number than the eight cases observed in the control group (P &#60; 0.05). In addition, there were no significant differences between the two groups in terms of seroma formation, mesh infection, recurrence of incisional hernia, the length of the postoperative hospital stay, and hospitalization expenses (Table 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64916/64916fig01.jpg" /> 
Figure 1: Measurement of the hernia ring defect size and marking the anti-adhesion mesh. (A) The size of the incisional hernia defect on the abdominal wall is measured. (B) The longitudinal axis length of the hernia ring defect is measured under laparoscopy. (C) The appropriate mesh size is selected. (D) The range of the vertical axis is marked on the mesh. (E) The fixation positions of the nails are marked on the edge of the mesh. (F) The fixation positions of the nails are marked in the middle of the mesh. This figure is reprinted with permission from Ning et al.10. <a href="https://www.jove.com/files/ftp_upload/64916/64916fig01large.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/64916/64916fig02.jpg" /> 
Figure 2: Steps of mesh placement under laparoscopy. (A) The layout of the puncture holes for an incisional hernia. (B)&#160;The mesh is placed into the abdominal cavity through the puncture hole. (C)&#160;The longitudinal axis of the mesh overlaps the longitudinal axis of the hernia ring defect (red dotted line). (D)&#160;The marked positions on the mesh longitudinal axis are fixed on the abdominal wall with non-absorbable nails (indicated by a red arrow). (E)&#160;The mesh is fixed to the abdominal wall along the marked points on the edge of the mesh (red arrow) with non-absorbable nails. (F)&#160;The mesh is fixed on the abdominal wall along the marked points in the middle of the mesh (red arrow) with absorbable nails. This figure is reprinted with permission from Ning et al.10. <a href="https://www.jove.com/files/ftp_upload/64916/64916fig02large.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/64916/64916fig03.jpg" /> 
Figure 3: Steps of the double-loop mesh fixation method. (A)After closing the hernia ring, the extent of the hernia ring defect is measured with a ruler under laparoscopy. (B) The appropriate mesh is selected according to the size of the hernia ring defect. (C) Fixation of the outer ring: the fixing nails are placed along the edge of the mesh to ensure that the distance between the hernia nail and the edge of the mesh is about 2-4 mm and the distance between the nails is about 2-3 cm. (D) Fixation of inner ring: the mesh is fixed about 2 cm along the outer edge of the hernia ring defect, and the distance between the nails is 3-5 cm. This figure is reprinted with permission from reference Ning et al.10. <a href="https://www.jove.com/files/ftp_upload/64916/64916fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64916/64916fig04.jpg" /> 
Figure 4: Comparison of the condition before and after the operation. (A)Photos of the incisional hernia of the abdominal wall before the operation. (B) A color ultrasound image of the abdominal wall before the operation. (C) A CT image of the abdominal wall before the operation. (D)&#160;Photos of the abdominal wall 3 months after the operation. (E) A color ultrasound image of the abdominal wall 1 month after the operation. (F) A&#160;CT image of the abdominal wall 3 months after the operation. <a href="https://www.jove.com/files/ftp_upload/64916/64916fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>experimental group 
			n=42</td>
			<td>control group 
			n=42</td>
			<td>P-value</td>
		</tr>
		<tr>
			<td>Age (years)</td>
			<td>52.5&#177;3.4</td>
			<td>53.2&#177;4.2</td>
			<td>0.4</td>
		</tr>
		<tr>
			<td>Gender (n) 
			Men 
			Women</td>
			<td> 
			26 
			16</td>
			<td> 
			27 
			15</td>
			<td>0.82</td>
		</tr>
		<tr>
			<td>BMI (kg/m2)</td>
			<td>24.0&#177;3.3</td>
			<td>23.5&#177;2.7</td>
			<td>0.45</td>
		</tr>
		<tr>
			<td>Disease time(yr)</td>
			<td>2.73&#177;0.51</td>
			<td>2.56&#177;0.64</td>
			<td>0.18</td>
		</tr>
		<tr>
			<td>Maximum defect 
			of hernia ring(cm)</td>
			<td></td>
			<td></td>
			<td>0.35</td>
		</tr>
		<tr>
			<td>&#8805;10cm</td>
			<td>11</td>
			<td>15</td>
			<td></td>
		</tr>
		<tr>
			<td>&#60;10cm</td>
			<td>31</td>
			<td>27</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1:&#160;Comparison of the baseline data of the experimental and control groups. The baseline characteristics compared included the patients&#39; age, sex, body mass index (BMI), the length of time of the hernia defect, and the maximum defect size of the hernia ring.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>experimental group 
			n=42</td>
			<td>control group 
			n=42&#160;&#160;</td>
			<td>P-value</td>
		</tr>
		<tr>
			<td>Mesh placement time (min)&#160;</td>
			<td>32.5&#177;11.6</td>
			<td>&#160;44.7&#177;12.5</td>
			<td>&#60;0.05</td>
		</tr>
		<tr>
			<td>Seroma</td>
			<td>3</td>
			<td>4</td>
			<td>1.00</td>
		</tr>
		<tr>
			<td>Mesh infection 
			Recurrence of hernia&#160;</td>
			<td>1 
			1</td>
			<td>2 
			3</td>
			<td>1.00 
			1.00</td>
		</tr>
		<tr>
			<td>Chronic pain</td>
			<td>2</td>
			<td>8</td>
			<td>&#160; &#60;0.05</td>
		</tr>
		<tr>
			<td>Postoperative hospital stay (days)</td>
			<td>5.8&#177;1.3</td>
			<td>&#160; 5.5&#177;1.1</td>
			<td>0.26</td>
		</tr>
		<tr>
			<td>Hospitalization cost (ten thousand )</td>
			<td>4.9&#177;0.6</td>
			<td>&#160;4.7&#177;0.5</td>
			<td>0.1</td>
		</tr>
	</tbody>
</table>
Table 2:&#160;Comparison of the intraoperative and postoperative data between the experimental and control groups. The indices compared between the two groups included placement time, seroma formation, mesh infection, hernia recurrence, chronic pain, the length of thepostoperative hospital stay, and the hospitalization costs.

Watch this Scientific Journal Video about Application of a New Mesh Fixation Method in Laparoscopic Incisional Hernia Repair at JoVE.com

Application of a New Mesh Fixation Method in Laparoscopic Incisional Hernia Repair

Presented here is a method for improving the mesh placement in laparoscopic incisional hernia repair, which can shorten the time required for mesh fixation and reduce the occurrence of postoperative chronic pain.

Laparoscopic incisional hernia repair using intraperitoneal onlay mesh (IPOM) is one of the most widely used minimally invasive methods for repairing ...

application-new-mesh-fixation-method-laparoscopic-incisional-hernia

Universidad San Sebastian

Research

JoVE Journal

Medicine

5.9K Views.  Sun Yat-sen University. Presented here is a method for improving the mesh placement in laparoscopic incisional hernia repair, which can shorten the time required for mesh fixation and reduce the occurrence of postoperative chronic pain.

Video: Application of a New Mesh Fixation Method in Laparoscopic Incisional Hernia Repair

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Transvaginal Mesh Insertion in the Ovine Model

This protocol describes mesh insertion into the rectovaginal septum in sheep using a single vaginal incision technique, with and without the trocar-guided insertion of anchoring arms. Parous sheep underwent the dissection of the rectovaginal septum, followed by the insertion of an implant with or without four anchoring arms, both designed to fit the ovine anatomy. The anchoring arms were put in place using a trocar and an "outside-in" technique. The cranial arms were passed through the obturator, gracilis, and adductor magnus muscles. The caudal arms were fixed near the sacrotuberous ligament, through the coccygeus muscles. This technique allows for the mimicking of surgical procedures performed in women suffering from pelvic organ prolapse. The anatomical spaces and elements are easily identified. The most critical part of the procedure is the insertion of the cranial trocar, which can easily penetrate the peritoneal cavity or the surrounding pelvic organs. This can be avoided by a more extensive retroperitoneal dissection and by guiding the trocar more laterally. This approach is designed only for experimental testing of novel implants in large animal models, as trocar-guided insertion is currently not used clinically.

This protocol describes mesh implantation in the ovine rectovaginal septum using a single vaginal incision technique, with and without the trocar-guided insertion of anchoring arms.

This protocol describes mesh insertion into the rectovaginal septum in sheep using a single vaginal incision technique, with and without the trocar-guided ...

Laparoscopic Repair of Para-Esophageal Hernia Using Absorbable Biosynthetic Mesh

Para-esophageal hernia repairs are challenging procedures and there is no consensus on the optimal approach to repair. Mesh reinforcement has been associated with lesser hernia recurrence when compared to the primary suture repair. The type of mesh that is most appropriate is still debatable. Synthetic and biosynthetic materials have been studied in the literature. It is well documented that a synthetic mesh is associated with esophageal erosion and migration into the stomach. Though there are limited long-term data on biosynthetic mesh, the short-term results are excellent and promising.
This paper illustrates how a biosynthetic prosthesis can be safely used with fibrin glue fixation and anterior Dor fundoplication to repair any para-esophageal defect. The absorbable biosynthetic mesh has been shown to produce good long-term patient satisfaction outcomes and low recurrence rates compared to conventional methods including repair with synthetic mesh. This technique also avoids the risk of esophageal erosion whilst strengthening the repair. Tacks that are still widely used to secure the mesh can be abandoned due to the associated risk of developing cardiac tamponade, or other disastrous consequences. This repair method, also, highlights how the prosthesis can be fashioned into a V-shape and easily placed in an onlay fashion behind the esophagus. The protocol demonstrates an alternative and safer method for mesh fixation using fibrin glue.

Presented here is a protocol of para-esophageal hernia repair. Use of absorbable biosynthetic mesh avoids the risk of erosion through the esophagus whilst reinforcing the repair. Glue fixation is preferred to avoid the risk of trauma such as bleeding or cardiac tamponade, which are associated with stitches or tacks.

Para-esophageal hernia repairs are challenging procedures and there is no consensus on the optimal approach to repair. Mesh reinforcement has been ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Application of Lucilia sericata Larvae in Debridement of Pressure Wounds in Outpatient Settings

Biological therapy using Lucilia sericata larvae has numerous advocates worldwide, yet it is still fairly unknown and not commonly applied in daily practice because of the limited awareness and insufficient experience of medical and nursing personnel. There are case reports suggesting that maggot therapy can be applied and supported by lay caregivers, provided they are supervised and informed by physicians/nurses. The foregoing observation suggests that the method should be considered for implementation by a wider group of caregivers if accepted and meticulously supervised by trained and experienced medical staff. The concerns related to the therapeutic use of maggots in certain regions seem understandable, but are not supported by scientific facts. It should be noted that many therapeutic agents (including brood) used in medicine are of natural origin, and are associated with low production costs and high possibilities of implementation in the course of therapy. By analyzing the literature and using our own clinical and research experience, we have come to conclusions related to using larvae therapy, as a quick and safe method providing cleaning and revitalization in the process of treating wounds of various etiologies, especially pressure ulcers. In the current study, medical-grade Lucilia sericata maggots were applied to remove necrotic tissue from deep pressure sores. The treatment is mostly accepted by both caregivers and patients. In most cases, it is conducted by trained and experienced medical personnel in home and outpatient settings. Over the course of the conducted analyzes involving the collected specimens, there were no statistically significant relationships (p&gt; 0.05) confirmed between the wound surface successfully cleared by brood and variables, such as time from wound formation, location, surface size, and the depth of damage to the tissue structure. The lack of statistical dependence may result from the small size of the studied group. Based on the current findings, we have formulated the following conclusions: Maggot Debridement Therapy (MDT) is a fast and effective method enabling the preparation of the wound bed. The use of MDT in outpatient settings is safe and acceptable for patients and their caregivers.

Application of Lucilia sericata Larvae in Debridement of Pressure Wounds in Outpatient Settings

Maggots of Lucilia sericata were used in the debridement of deep pressure sores in outpatient settings for the treatment of pressure sores involving the complete skin thickness. The technique presented in the article does not pose risks for the patient.

Biological therapy using Lucilia sericata larvae has numerous advocates worldwide, yet it is still fairly unknown and not commonly applied in daily ...

Laparoscopic Non-Mesh Cerclage Pectopexy for Pelvic Organ Prolapse

Pelvic organ prolapse (POP) is widespread among the female population and significantly impairs the patient's quality of life. It is important to restore apical support for treating POP. Sacrocolpopexy and pectopexy are indicated for apical prolapse. Using a synthetic mesh in these techniques increases success by enhancing apical support. However, the implantation of synthetic mesh is associated with mesh-related complications. In addition, the exorbitant cost of synthetic mesh and lack of universal access limit the popularity of these procedures. The current study develops a unique technique known as laparoscopic non-mesh cerclage pectopexy (LNMCP), in which permanent cervical cerclage sutures are embedded in the round ligament until the iliopectineal ligament. The iliopectineal ligament was sutured, resulting in a firm cervical suspension. The procedure was successfully performed in 16 cases in the hospital. The surgical duration was 67.8 min &plusmn; 15.5 min, and the blood loss was 73.1 mL &plusmn; 51.1 mL. No procedural complications were seen. LNMCP is associated with an objective success rate of 100% and a subjective success rate of 93.8%. LNMCP for patients with apical prolapse obviates the need for a mesh, thereby avoiding complications associated with mesh erosion and reducing medical costs. In addition, it is easy to perform even in resource-poor areas without access to synthetic mesh.

The present pilot study describes the development of laparoscopic non-mesh cerclage pectopexy to treat pelvic organ prolapse. The procedure can be used to prevent any complications associated with the use of mesh.

Pelvic organ prolapse (POP) is widespread among the female population and significantly impairs the patient's quality of life. It is important to restore ...