Comparison of Surgical and Cadaveric Intestine as a Source of Crypt Culture in Humans.

Human small intestinal crypts are the source of intestinal stem cells (ISCs) that are capable of undergoing self-renewal and differentiation to an epithelial layer. The development of methods to expand the ISCs has provided opportunities to model human intestinal epithelial disorders. Human crypt samples are usually obtained from either endoscopic or discarded surgical samples, and are thereby exposed to warm ischemia, which may impair their in vitro growth as three-dimensional culture as spheroids or enteroids. In this study we compared duodenal samples obtained from discarded surgical samples to those isolated from whole-body preserved cadaveric donors to generate in vitro cultures. We also examined the effect of storage solution (phosphate-buffered saline or University of Wisconsin [UW] solution) as well as multiple storage times on crypt isolation and growth in culture. We found that intestinal crypts were successfully isolated from cadaveric tissue stored for up to 144 h post-procurement and also were able to generate enteroids and spheroids in certain media conditions. Surgical samples stored in UW after procurement were sufficiently viable up to 24 h and also allowed the generation of enteroids and spheroids. We conclude that surgical samples stored for up to 24 h post-procurement in UW solution allowed for delayed crypt isolation and viable in vitro cultures. Furthermore, in situ, hypothermic preservation in cadaveric duodenal samples permitted crypt/ISC isolation, and successful culture of spheroids and enteroids from tissues held for up to 6 days post-procurement.

Intestinal epithelial replacement by transplantation of cultured murine and human cells into the small intestine.

Adult intestinal epithelial stem cells are a promising resource for treatment of intestinal epithelial disorders that cause intestinal failure and for intestinal tissue engineering. We developed two different animal models to study the implantation of cultured murine and human intestinal epithelial cells in the less differentiated "spheroid" state and the more differentiated "enteroid" state into the denuded small intestine of mice. Engraftment of donor cells could not be achieved while the recipient intestine remained in continuity. However, we were able to demonstrate successful implantation of murine and human epithelial cells when the graft segment was in a bypassed loop of jejunum. Implantation of donor cells occurred in a random fashion in villus and crypt areas. Engraftment was observed in 75% of recipients for murine and 36% of recipients for human cells. Engrafted spheroid cells differentiated into the full complement of intestinal epithelial cells. These findings demonstrate for the first time successful engraftment into the small bowel which is optimized in a bypassed loop surgical model.

Three-dimensionally printed surface features to anchor endoluminal spring for distraction enterogenesis.

Spring-mediated distraction enterogenesis has been studied as a novel treatment for short bowel syndrome (SBS). Previous approaches are limited by multiple surgeries to restore intestinal continuity. Purely endoluminal devices require a period of intestinal attachment for enterogenesis. The purpose of this study is to modify the device to prevent premature spring migration in a porcine model. Two models were created in juvenile mini-Yucatan pigs for the placement of three-dimensionally printed springs. (1) Two Roux-en-y jejunojenostomies with two Roux limbs were made. A spring with bidirectional hooked surface features was placed in one Roux limb and a spring with smooth surface was placed in the other Roux limb. (2) The in-continuity model had both hooked and smooth surface springs placed directly in intestinal continuity. Spring location was evaluated by weekly radiographs, and the intestine was retrieved after 2 to 4 weeks. Springs with smooth surfaces migrated between 1 to 3 weeks after placement in both porcine models. Springs with bidirectional hooked surface features were anchored to the intestine for up to 4 weeks without migration. Histologically, the jejunal architecture showed significantly increased crypt depth and muscularis thickness compared to normal jejunum. Bidirectional features printed on springs prevented the premature migration of endoluminal springs. These novel spring anchors allowed for their endoluminal placement without any sutures. This approach may lead to the endoscopic placement of the device for patients with SBS.

Subcutaneous cefazolin to reduce surgical site infections in a porcine model.

BACKGROUND: Surgical site infections (SSIs) pose a significant health and financial burden. A key aspect of appropriate prophylaxis is the administration of antibiotics intravenously (IV). However, subcutaneous administration of antibiotics is not well described in the literature. During surgery, we hypothesize that subcutaneous injection may provide better protection against SSIs. To better understand the kinetics after subcutaneous injection, we describe the serum concentrations of cefazolin in a porcine model. MATERIALS AND METHODS: Juvenile mini-Yucatan pigs were administered 20 mL of 25 mg/kg cefazolin subcutaneously, and serial blood samples were taken for 3 h. Blood samples were analyzed for cefazolin concentration using chromatography. Pharmacokinetic data were calculated based on the blood serum concentrations. RESULTS: Maximum serum concentrations of cefazolin were achieved 42.6 ± 2.0 min after the time of injection and were found to be 18.8 ± 7.4 µg/mL. The elimination rate constant was 0.0033 ± 0.0016 min-1 and the half-life was 266 ± 149 min. The area under the curve was 4940 ± 1030 µg × min/mL. The relative bioavailability of subcutaneous injection was 95% +5%/-20%. CONCLUSIONS: Subcutaneous administration of cefazolin achieves a significantly lower maximum serum concentration than IV injection. As a result, higher doses of antibiotic can be injected locally without incurring systemic toxicity. Subcutaneous administration will therefore result in higher concentrations of antibiotic for a longer time at the incision site compared with standard IV administration. This strategy of antibiotic delivery may be more effective in preventing SSIs. Further studies are needed to detail the exact effect of subcutaneous antibiotic injection on SSI rates.

Re-exploration of vertical rectus abdominis myocutaneous flap for vaginal reconstruction: Case report and review of the literature.

The vertical rectus abdominis myocutaneous (VRAM) flap is a versatile and well-established reconstructive technique for many defects created as a result of colorectal and gynecologic extirpation. However, major re-operation in the pelvis following a VRAM flap reconstruction several months later is uncommon, and the safety and integrity of the VRAM flap in this setting has not been described. This case examines VRAM flap preservation during repeat exploratory laparotomy, and a unique view of the VRAM flap during interval exploration. We demonstrate an intact flap after lysis of adhesions with an audible Doppler signal, and maintenance of flap integrity in the postoperative period. This further substantiates its use as a durable rotational flap for perineal tissue defects.

Feasibility and scalability of spring parameters in distraction enterogenesis in a murine model.

BACKGROUND: Distraction enterogenesis has been investigated as a novel treatment for short bowel syndrome (SBS). With variable intestinal sizes, it is critical to determine safe, translatable spring characteristics in differently sized animal models before clinical use. Nitinol springs have been shown to lengthen intestines in rats and pigs. Here, we show spring-mediated intestinal lengthening is scalable and feasible in a murine model. MATERIALS AND METHODS: A 10-mm nitinol spring was compressed to 3 mm and placed in a 5-mm intestinal segment isolated from continuity in mice. A noncompressed spring placed in a similar fashion served as a control. Spring parameters were proportionally extrapolated from previous spring parameters to accommodate the smaller size of murine intestines. After 2-3 wk, the intestinal segments were examined for size and histology. RESULTS: Experimental group with spring constants, k = 0.2-1.4 N/m, showed intestinal lengthening from 5.0 ± 0.6 mm to 9.5 ± 0.8 mm (P < 0.0001), whereas control segments lengthened from 5.3 ± 0.5 mm to 6.4 ± 1.0 mm (P < 0.02). Diameter increased similarly in both groups. Isolated segment perforation was noted when k ≥ 0.8 N/m. Histologically, lengthened segments had increased muscularis thickness and crypt depth in comparison to normal intestine. CONCLUSIONS: Nitinol springs with k ≤ 0.4 N/m can safely yield nearly 2-fold distraction enterogenesis in length and diameter in a scalable mouse model. Not only does this study derive the safe ranges and translatable spring characteristics in a scalable murine model for patients with short bowel syndrome, it also demonstrates the feasibility of spring-mediated intestinal lengthening in a mouse, which can be used to study underlying mechanisms in the future.

New insights and interventions for short bowel syndrome.

PURPOSE OF REVIEW: This review summarizes recent innovations in the treatment of patients with short bowel syndrome. RECENT FINDINGS: The use of surgical procedures, growth factor stimulation, and bioengineering approaches to increase absorptive surface area of the intestine is examined. While the morphology of the intestine is clearly altered by these interventions, it is less clear that the overall function of the intestine is improved. SUMMARY: Continued innovations will likely bring about new therapeutic options for patients with short bowel syndrome. Careful evaluations of the impact of these interventions await controlled clinical trials.

Malpractice in Cases of Pediatric Appendicitis.

Appendicitis is one of the most common diagnoses in children and is frequently the focus of alleged malpractice. Causes for medical malpractice claims and outcomes of disputes in pediatric patients with appendicitis are currently unknown. A retrospective database review of all medical malpractice claims concerning the diagnosis of appendicitis from 1984 to 2013 in pediatric patients was performed. Alleged claims, causes of malpractice, and outcomes were recorded and analyzed. Of the 203 included cases, failure or delays in diagnosing appendicitis are the most common causes of malpractice lawsuits and account for the majority of the largest payments to plaintiffs outcomes. Cases that ultimately went to trial resulted in defense verdicts in 67.5%. Mortality occurred in 19.9% of included cases. Timely diagnosis of appendicitis in children should be the focus of physicians across all specialties to improve patient safety and potentially reduce medicolegal liability.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Toll-like receptor 4 ablation improves stem cell survival after hypoxic injury.

INTRODUCTION: Mesenchymal stem cell (MSC) therapy improves cardiac function after ischemia/reperfusion injury, but its effectiveness is limited by MSC survival in hypoxic environments. Toll-like receptor 4 (TLR4) contributes to pro-apoptotic signaling under hypoxic conditions. Activation of intracellular AKT and ERK pathways opposes this signal and improves cell survival. It is unknown whether ablation of TLR4 affects these pathways after hypoxic injury in MSCs. We hypothesized that: 1) TLR4 knockout (TLR4KO) in MSCs improves survival after hypoxic injury; and 2) this survival difference is due to improved signaling in the AKT and ERK pathways. MATERIALS AND METHODS: Murine wild-type (WT) and TLR4KO MSCs were harvested from bone marrow and grown in vitro. A total of 0.1 × 10(6) cells/well were incubated in hypoxic conditions versus normoxic controls. After 24 h, these groups were examined for cell survival via counting and compared using a t-test with P < 0.05 = statistical significance. AKT and ERK concentrations were measured in lysate using Western blot analysis. RESULTS: The morphology of WT and TLR4KO MSCs was similar. In line with our previous findings, hypoxia did significantly increase cell death in WT cells (1.79 × 10(5) living cells/mL control versus 0.88 × 10(5) hypoxia, P < 0.05). Hypoxic injury did not increase cell death in the TLR4KO group (1.68 × 10(5) control versus 1.82 × 10(5) hypoxia, P < 0.05). Increased AKT activation was observed in all TLR4KO groups. TLR4 did not affect phosphorylated ERK levels. CONCLUSION: TLR4-knockout MSCs show improved survival after hypoxic injury because of increased AKT pathway signal. Use of TLR4-knockout MSCs in ischemia/reperfusion studies results in enhanced cardioprotection; improved stem cell survival was likely a contributing factor.