Joshua D. Rouch

Development of Functional Microfold (M) Cells from Intestinal Stem Cells in Primary Human Enteroids

Background & Aims Intestinal microfold (M) cells are specialized epithelial cells that act as gatekeepers of luminal antigens in the intestinal tract. They play a critical role in the intestinal mucosal immune response through transport of viruses, bacteria and other particles and antigens across the epithelium to immune cells within Peyer’s patch regions and other mucosal sites. Recent studies in mice have demonstrated that M cells are generated from Lgr5+ intestinal stem cells (ISCs), and that infection with Salmonella enterica serovar Typhimurium increases M cell formation. However, it is not known whether and how these findings apply to primary human small intestinal epithelium propagated in an in vitro setting. Methods Human intestinal crypts were grown as monolayers with growth factors and treated with recombinant RANKL, and assessed for mRNA transcripts, immunofluorescence and uptake of microparticles and S. Typhimurium. Results Functional M cells were generated by short-term culture of freshly isolated human intestinal crypts in a dose- and time-dependent fashion. RANKL stimulation of the monolayer cultures caused dramatic induction of the M cell-specific markers, SPIB, and Glycoprotein-2 (GP2) in a process primed by canonical WNT signaling. Confocal microscopy demonstrated a pseudopod phenotype of GP2-positive M cells that preferentially take up microparticles. Furthermore, infection of the M cell-enriched cultures with the M cell-tropic enteric pathogen, S. Typhimurium, led to preferential association of the bacteria with M cells, particularly at lower inoculum sizes. Larger inocula caused rapid induction of M cells. Conclusions Human intestinal crypts containing ISCs can be cultured and differentiate into an epithelial layer with functional M cells with characteristic morphological and functional properties. This study is the first to demonstrate that M cells can be induced to form from primary human intestinal epithelium, and that S. Typhimurium preferentially infect these cells in an in vitro setting. We anticipate that this model can be used to generate large numbers of M cells for further functional studies of these key cells of intestinal immune induction and their impact on controlling enteric pathogens and the intestinal microbiome.

Long-term renewable human intestinal epithelial stem cells as monolayers: A potential for clinical use.

PURPOSE Current culture schema for human intestinal stem cells (hISCs) frequently rely on a 3D culture system using Matrigel™, a laminin-rich matrix derived from murine sarcoma that is not suitable for clinical use. We have developed a novel 2D culture system for the in vitro expansion of hISCs as an intestinal epithelial monolayer without the use of Matrigel. METHODS Cadaveric duodenal samples were processed to isolate intestinal crypts from the mucosa. Crypts were cultured on a thin coat of type I collagen or laminin. Intestinal epithelial monolayers were supported with growth factors to promote self-renewal or differentiation of the hISCs. Proliferating monolayers were sub-cultured every 4-5days. RESULTS Intestinal epithelial monolayers were capable of long-term cell renewal. Less differentiated monolayers expressed high levels of gene marker LGR5, while more differentiated monolayers had higher expressions of CDX2, MUC2, LYZ, DEF5, and CHGA. Furthermore, monolayers were capable of passaging into a 3D culture system to generate spheroids and enteroids. CONCLUSION This 2D system is an important step to expand hISCs for further experimental studies and for clinical cell transplantation. LEVEL OF EVIDENCE 1 Experimental.

Repeated Mechanical Lengthening of Intestinal Segments in a Novel Model

PURPOSE Currently, animal models used for mechanical intestinal lengthening utilize a single lengthening procedure prior to analysis or restoration back into continuity. Here we developed a novel surgical model to examine the feasibility of repeated lengthening of intestinal segments. METHODS A Roux-en-Y jejunojejunostomy with a blind Roux limb was created in rats. An encapsulated polycaprolactone spring was placed into a 1cm segment of the Roux limb. After 4 weeks, a second encapsulated PCL spring was inserted into a 1cm portion of the lengthened segment. After another 4 weeks, the repeatedly lengthened segments were retrieved for histological analyses. RESULTS Jejunal segments of the Roux limb were successfully lengthened from 1.0 cm to 2.6 ± 0.7 cm. Four weeks after the second PCL spring placement, 1.0 cm of the previously lengthened segment increased to 2.7 ± 0.8 cm. Stronger mechanical force was required to achieve subsequent re-lengthening. Lengthened and re-lengthened segments had increased smooth muscle thickness and crypt depth when compared to normal jejunal mucosa. CONCLUSION Using the Roux-en-Y model, previously lengthened segments of intestine can be successfully re-lengthened. Intestinal segments may be subjected to multiple lengthening procedures to achieve clinically significant length for the treatment of short bowel syndrome.

Same-Day Discharge in Laparoscopic Acute Non-Perforated Appendectomy

BACKGROUND Small studies done during the past decade have demonstrated same-day discharge after appendectomy as an option for non-perforated appendicitis. Here we have examined a large cohort to confirm that same-day discharge in acute non-perforated appendicitis is a safe option. STUDY DESIGN This was a retrospective study of patients from 14 Southern California Region Kaiser Permanente medical centers. All patients older than 18 years of age with acute, non-perforated appendicitis who underwent a laparoscopic appendectomy between 2010 and 2014 were included. We compared patients discharged on the day of surgery with patients hospitalized for 1 night. We examined readmission rates, complication rates, postoperative emergency department visits, postoperative diagnostic or therapeutic radiology visits, reoperations, and cost of treatment. RESULTS The cohort was composed of 12,703 patients; 6,710 patients were in the same-day discharge group and 5,993 patients were in the hospitalized group. Patients in the same-day discharge group had a lower rate of readmission within 30 days when compared with the hospitalized group (2.2% vs 3.1%; p < 0.005). In both groups, postoperative rates of visits to emergency or radiology department for diagnostic or therapeutic imaging studies were statistically similar. Postoperative general surgery department visits were slightly higher in the hospitalized group (85% vs 81%; p < 0.001). CONCLUSIONS Adult patients with acute, non-perforated appendicitis can be discharged safely on the day of surgery without higher rates of postoperative complication or readmission rates compared with those hospitalized after surgery. In addition, same-day discharge in this patient group is cost-effective.

Scalability of an endoluminal spring for distraction enterogenesis

INTRODUCTION Techniques of distraction enterogenesis have been explored to provide increased intestinal length to treat short bowel syndrome (SBS). Self-expanding, polycaprolactone (PCL) springs have been shown to lengthen bowel in small animal models. Their feasibility in larger animal models is a critical step before clinical use. METHODS Juvenile mini-Yucatan pigs underwent jejunal isolation or blind ending Roux-en-y jejunojejunostomy with insertion of either a PCL spring or a sham PCL tube. Extrapolated from our spring characteristics in rodents, proportional increases in spring constant and size were made for porcine intestine. RESULTS Jejunal segments with 7mm springs with k between 9 and 15N/m demonstrated significantly increased lengthening in isolated segment and Roux-en-y models. Complications were noted in only two animals, both using high spring constant k>17N/m. Histologically, lengthened segments in the isolated and Roux models demonstrated significantly increased muscularis thickness and crypt depth. Restoration of lengthened, isolated segments back into continuity was technically feasible after 6weeks. CONCLUSION Self-expanding, endoluminal PCL springs, which exert up to 0.6N force, safely achieve significant intestinal lengthening in a translatable, large-animal model. These spring characteristics may provide a scalable model for the treatment of SBS in children.

Spring-mediated distraction enterogenesis in-continuity

PURPOSE Distraction enterogenesis has been investigated as a novel treatment for patients with short bowel syndrome (SBS) but has been limited by loss of intestinal length during restoration and need for multiple bowel surgeries. The feasibility of in-continuity, spring-mediated intestinal lengthening has yet to be demonstrated. METHODS Juvenile mini-Yucatan pigs underwent in-continuity placement of polycaprolactone (PCL) degradable springs within jejunum. Methods used to anchor the spring ends to the intestine included full-thickness sutures and a high-friction surface spring. Spring constant (k) was 6-15N/m. Bowel was examined for length and presence of spring at 1 to 4weeks. RESULTS Animals tolerated in-continuity lengthening without bowel obstruction for up to 29days. In-continuity jejunum with springs demonstrated intestinal lengthening by 1.47-fold ±0.11. Five springs had detached prematurely, and lengthening could not be assessed. Histologically, in-continuity jejunum showed significantly increased crypt depth and muscularis thickness in comparison to normal jejunum. CONCLUSION Self-expanding endoluminal springs placed in continuity could lengthen intestine without obstruction in a porcine model. This is the first study showing safety and efficacy of a self-expanding endoluminal device for distraction enterogenesis. This is proof-of-concept that in-continuity spring lengthening is feasible and demonstrates its therapeutic potential in SBS. LEVEL OF EVIDENCE Level 3.

Mechanical lengthening in multiple intestinal segments in-series

PURPOSE Current models of mechanical intestinal lengthening employ a single device in an isolated segment. Here we demonstrate that polycaprolactone (PCL) springs can be deployed in-series to lengthen multiple intestinal segments simultaneously to further increase overall intestinal length. METHODS A Roux-en-y jejunojejunostomy with a blind Roux limb was created in the proximal jejunum of rats. Two encapsulated 10-mm PCL springs were placed in-series into the Roux limb and were secured with clips. After 4weeks, the lengthened segments were retrieved for histological analyses. RESULTS Lengthening two intestinal segments simultaneously was achieved by placing two PCL springs in-series. The total combined length of the lengthened segments in-series was 45±4mm. The two jejunal segments with PCL springs (25±2 and 20±2mm) were significantly longer than control segments without the spring (14±1mm, p<0.05). CONCLUSION Spring-mediated lengthening can be achieved using multiple springs placed sequentially. The use of the Roux-en-y surgical model allowed easy insertion of springs in a blind Roux limb and arrange them in-series. Combined with relengthening techniques, we can use these methods to increase the length of small intestine to reach clinical significance. LEVEL OF EVIDENCE 1 Experimental.

Short- and Long-term Results of Unroofing and Marsupialization for Adolescent Pilonidal Disease

Shortand Long-term Results of Unroofing and Marsupialization for Adolescent Pilonidal Disease Pilonidal disease is a benign and often chronic condition of the skin/soft tissue in the sacrococcygeal region. For failed response to nonoperative management, there is currently no consensus among a variety of different operative techniques.1,2 Currently, wide local excision (WLE) with or without closure is advocated by many.3,4 This technique is characterized by wide excision of all involved tissue. This is followed by operative closure or is allowed to heal by secondary intention. These therapies can result in significant morbidity, long healing times, and high risk for recurrence. Flap techniques are durable but difficult to learn and require drains and/or prolonged immobilization.5 Previously, we reported outcomes following unroofing and marsupialization (UM).6 This simple technique involves incision of the sinus tract and marsupialization of the skin edges using absorbable suture. No normal skin tissue is excised in this technique. The purpose of this study is to report our current experience with UM vs WLE as surgical therapy for pilonidal disease and provide, to our knowledge, the first report of long-term follow-up data for UM.

Basic fibroblast growth factor eluting microspheres enhance distraction enterogenesis.

PURPOSE The purpose of this study was to determine if distraction enterogenesis using self-expanding polycaprolactone (PCL) springs is a potential therapy for short bowel syndrome. Sustained release basic fibroblast growth factor (bFGF) microspheres have been shown to induce angiogenesis and intestinal regeneration in tissue engineered scaffolds. We hypothesized that the provision of bFGF-loaded microspheres would increase angiogenesis and thereby enhance the process of enterogenesis. METHODS A 10-mm segment of rodent jejunum was isolated and an encapsulated PCL spring inserted. Blank or bFGF-loaded microspheres were delivered to the segment. After 4weeks, jejunal segments were assessed for lengthening, morphology, quantification of blood vessels, and ganglia. RESULTS Lengthened intestinal segments receiving bFGF microspheres demonstrated significantly increased microvascular density compared to those with blank microspheres. There were also significantly more submucosal and myenteric ganglia in the segments that received bFGF microspheres. Segments achieved similar lengthening and final muscular thickness in both blank and bFGF groups, but the bFGF microsphere caused a significant increase in luminal diameter of the jejunal segment. CONCLUSION Sustained release bFGF microspheres enhanced distraction enterogenesis through improved vascularity. The synergy of growth factors such as bFGF with distraction enterogenesis may yield improved results for the future treatment of patients with short bowel syndrome.

Innovation in Pediatric Surgical Education for General Surgery Residents: A Mobile Web Resource

BACKGROUND/OBJECTIVES General surgery residents lack a standardized educational experience in pediatric surgery. We hypothesized that the development of a mobile educational interface would provide general surgery residents broader access to pediatric surgical education materials. METHODS We created an educational mobile website for general surgery residents rotating on pediatric surgery, which included a curriculum, multimedia resources, the Operative Performance Rating Scale (OPRS), and Twitter functionality. Residents were instructed to consult the curriculum. Residents and faculty posted media using the Twitter hashtag, #UCLAPedSurg, and following each surgical procedure reviewed performance via the OPRS. Site visits, Twitter posts, and OPRS submissions were quantified from September 2013 to July 2014. RESULTS The pediatric surgery mobile website received 257 hits; 108 to the homepage, 107 to multimedia, 28 to the syllabus, and 19 to the OPRS. All eligible residents accessed the content. The Twitter hashtag, #UCLAPedSurg, was assigned to 20 posts; the overall audience reach was 85 individuals. Participants in the mobile OPRS included 11 general surgery residents and 4 pediatric surgery faculty. CONCLUSION Pediatric surgical education resources and operative performance evaluations are effectively administered to general surgery residents via a structured mobile platform.