Veronica F. Sullins

A novel biodegradable device for intestinal lengthening

PURPOSE Previous studies demonstrated successful mechanical lengthening of rat jejunum using an encapsulated Nitinol spring device over a stabilizing guidewire. We sought to improve the applicability of intestinal lengthening by creating a biodegradable device. METHODS Using properties of the Nitinol spring device, polycaprolactone (PCL) springs with similar outer diameter and spring constant were created. After in vitro testing in dry and hydrated environments, they were used to lengthen 1-cm isolated segments of rat jejunum in vivo. Retrieved segments were analyzed histologically. RESULTS Optimal PCL spring devices had an average spring constant 1.8 ± 0.4 N/m, pitch 1.55 ± 0.85 mm, and band width 0.825 ± 0.016 mm. In vitro testing demonstrated stable spring constants. Jejunal segments were lengthened from 1.0 cm to 2.7 ± 0.4 cm without needing a stabilizing guidewire. Histology demonstrated increased smooth muscle thickness and fewer ganglia compared to controls. Lengthened jejunum was successfully restored into intestinal continuity and demonstrated peristalsis under fluoroscopy. CONCLUSIONS A novel biodegradable spring device was successfully created and used to mechanically lengthen intestinal segments. Use of a biodegradable device may obviate the need for retrieval after lengthening. This improves device applicability and may be useful for the treatment of short bowel syndrome.

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.

Function of mechanically lengthened jejunum after restoration into continuity

PURPOSE Distraction enterogenesis is a potential treatment for patients with short bowel syndrome. We previously demonstrated successful lengthening of jejunum using a degradable spring device in rats. Absorptive function of the lengthened jejunum after restoration into intestinal continuity needs to be determined. METHODS Encapsulated polycaprolactone springs were placed into isolated jejunal segments in rats for four weeks. Lengthened segments of jejunum were subsequently restored into intestinal continuity. Absorption studies were performed by placing a mixture of a non-absorbable substrate and glucose into the lumen of the restored jejunum. RESULTS Restored jejunal segments demonstrated visible peristalsis at specimen retrieval. Compared to normal jejunal controls, restored segments demonstrated equal water absorption and greater glucose absorption. Restored segments had thicker smooth muscle, increased villus height, increased crypt depth, and decreased sucrase activity compared to normal jejunum. The density of enteric ganglia increased after restoration to near normal levels in the submucosa and to normal levels in the myenteric plexus. CONCLUSION Jejunum lengthened with a degradable device demonstrates peristaltic and enzymatic activity as well as glucose and water absorption after restoration into intestinal continuity. Our findings further demonstrate the therapeutic potential of a degradable device.

Intestinal lengthening in an innovative rodent surgical model

PURPOSE Current animal models of mechanical lengthening separate intestinal segments from enteric continuity. Such models are difficult to use for repeated lengthening procedures and result in intestinal tissue loss during restoration into continuity. We sought to create a novel surgical model to allow multiple lengthening procedures for the purpose of maximizing the net increase in tissue after intestinal lengthening. METHODS A Roux-en-y jejunojejunostomy with a 6-cm blind-ended Roux limb was created in the proximal jejunum of rats. Encapsulated 1-cm polycaprolactone springs were placed into the closed end of the roux limb and secured with a vessel loop. After 4weeks, lengthened segments and normal jejunum were retrieved for histologic analysis. RESULTS Jejunal segments were lengthened from 1.0cm to 3.0cm. Lengthened segments had increased smooth muscle thickness, fewer submucosal ganglia, and similar numbers of myenteric ganglia compared to normal intestine. When compared to normal jejunal mucosa, lengthened segments demonstrated unchanged villus height and increased crypt depth. CONCLUSIONS We created an innovative surgical model for intestinal lengthening and successfully lengthened jejunal segments with a degradable spring. The Roux-en-y model may allow the use of a degradable spring for the treatment of short bowel syndrome.

Transplanted skin-derived precursor stem cells generate enteric ganglion-like structures in vivo

INTRODUCTION Hirschsprungs disease is characterized by a developmental arrest of neural crest cell migration, causing distal aganglionosis. Transplanted cells derived from the neural crest may regenerate enteric ganglia in this condition. We investigated the potential of skin-derived precursor cells (SKPs) to engraft and to differentiate into enteric ganglia in aganglionic rat intestine in vivo. METHODS Adult Lewis rat jejunal segments were separated from intestinal continuity and treated with benzalkonium chloride to induce aganglionosis. Ganglia were identified via immunohistochemical stains for S100 and β-III tubulin (TUJ1). SKPs were procured from neonatal Lewis rats expressing enhanced green fluorescent protein (GFP) and cultured in neuroglial-selective media. SKP cell line expansion was quantified, and immunophenotypes were assessed by immunocytochemistry. Aganglionic segments underwent SKP transplantation 21-79days after benzalkonium chloride treatment. The presence of GFP+cells, mature neurons, and mature glia was evaluated at posttransplant days 1, 6, and 9. RESULTS Benzalkonium chloride-induced aganglionosis persisted for at least 85days. Prior to differentiation, SKPs expressed S100, denoting neural crest lineage, and nestin, a marker of neuronal precursors. Differentiated SKPs in vitro expressed GFAP, a marker of glial differentiation, as well as TUJ1 and several enteric neurotransmitters. After transplantation, GFP+structures resembling ganglia were identified between longitudinal and circular smooth muscle layers. CONCLUSION SKPs are capable of engraftment, migration, and differentiation within aganglionic rodent intestine in vivo. Differentiated SKPs generate structures that resemble enteric ganglia. Our observations suggest that SKPs represent a potential gangliogenic therapeutic agent for Hirschsprungs disease.

A novel method of esophageal lengthening in a large animal model of long gap esophageal atresia.

PURPOSE Long gap esophageal atresia remains a significant treatment challenge. We aimed to create the first large animal model of long gap esophageal atresia to test a degradable esophageal lengthening device. METHODS The distal esophagus was divided 2 cm above the gastroesophageal junction in 6 minipigs. A polycaprolactone (PCL) spring device was secured inside the distal esophageal segment, and the end was oversewn. Nonexpanding PCL tubes served as controls. An esophagogastric anastomosis was created to restore continuity. After 4 weeks, the distal esophageal pouch was analyzed. RESULTS The distal esophageal pouch of experimental animals increased in length from 1.9 to 4.5 cm. Control animals demonstrated no change. When comparing lengthened to native esophagus, there was no difference in the thickness of muscularis mucosa or muscularis propria. Mechanically lengthened esophagus showed mild to moderate superficial inflammation and fibrosis. There were no differences in the number of myenteric or submucosal ganglia. CONCLUSION We created the first porcine model of long gap esophageal atresia and lengthened the distal esophagus with an internally placed device. This model may be used to explore novel therapies in the management of long gap esophageal atresia.

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.

A novel in vivo model of permanent intestinal aganglionosis

BACKGROUND Enteric neuromuscular disease is a characteristic of several disease states, including Hirschsprung disease, esophageal achalasia, Chagas disease, and gastroparesis. Medical therapy for these conditions is limited, and surgical intervention may incur significant morbidity. Alternatively, transplantation of neural progenitor cells may regenerate enteric ganglia. Existing aganglionosis model systems are limited by swift animal demise or by spontaneous regeneration of native ganglia. We propose a novel protocol to induce permanent aganglionosis in a segment of rat jejunum, which may serve as an experimental transplantation target for cellular therapy. MATERIALS AND METHODS This protocol was performed in 17 adult female Sprague-Dawley rats. A laparotomy was performed and a 1-cm segment of jejunum was isolated from continuity. Among 14 rats, the isolated segments were treated with benzalkonium chloride (BAC) for 20 min to induce aganglionosis. Jejunal segment isolation was performed without BAC treatment in three rats. The animals were euthanized at posttreatment days 21-166. Muscle layer diameter was compared among normal, isolated, and BAC-treated isolated jejunal segments. The presence of jejunal ganglia was documented by immunohistochemical staining (IHC) for beta-III tubulin (TUJ1) and S100, markers of neuronal and glial cell lineages, respectively. RESULTS Ganglia were identified by IHC in normal and isolated jejunal segments. Isolated segments had significantly hypertrophied smooth muscle layers compared with normal jejunum (diameter 343 ± 53 μm versus 211 ± 37 μm, P < 0.0001). BAC-treated jejunal segments had no IHC evidence of ganglionic structures. Aganglionosis was persistent in all specimens up to 166 days after treatment. CONCLUSIONS The exclusion of a jejunal segment from continuity and concurrent treatment with BAC results in an effective, reproducible, and permanent model of aganglionosis. Muscular hypertrophy and aganglionosis in the isolated jejunal segment make it an ideal recipient site for transplantation of neuroglial precursor cells.

Skin-derived precursors generate enteric-type neurons in aganglionic jejunum

PURPOSE Skin-derived precursor cells (SKPs) may regenerate the enteric nervous system in Hirschsprungs disease. SKPs migrate and differentiate into myenteric ganglia in aganglionic intestine. We sought to characterize the time-course of SKP gangliogenesis and enteric neurotransmitter synthesis in vivo. METHODS Adult Lewis rat jejunal segments were isolated and denervated with benzalkonium chloride (BAC). Denervation was evaluated by immunohistochemical (IHC) stains for markers of mature neuronal and glial cells. Green fluorescent protein (GFP)-expressing neonatal rat SKPs were cultured in neuroglial-selective medium. SKPs were transplanted into aganglionic segments 65-85days after BAC treatment. IHC was performed to identify glia, neurons, and neurotransmitter synthesis in GFP+cells between post-transplant days 1 and 28. RESULTS Aganglionosis was confirmed by IHC. On post-transplant days 1 and 2, GFP+cells were detected near injection sites within the muscularis propria. GFP+cell clusters were evident only between longitudinal and circular smooth muscle layers at post-transplant days 14, 21, and 28. These structures co-expressed markers of mature neurons and gliocytes. Several markers of neurotransmitter synthesis were detected in GFP+clusters at days 21 and 28. CONCLUSION SKPs are capable of enteric neuroglial differentiation in vivo. SKPs migrate to the intermuscular layer of aganglionic intestine within days of transplantation. Our observations suggest that SKPs are capable of generating enteric ganglia in aganglionic intestine.

A durable model of Hirschsprung's colon.

INTRODUCTION Hirschsprungs disease is characterized by colonic aganglionosis, curable only by surgical correction. Stem cells may offer regenerative benefits while preventing surgical risks. Existing Hirschsprungs model systems are limited by alimentary compromise and spontaneous ganglionic reconstitution. We endeavored to generate a model of permanent colonic aganglionosis to support longitudinal cell therapy studies. METHODS Among adult female Lewis rats (n=11), laparotomy was performed and one-centimeter segments of descending colon were isolated from continuity and denervated by trans-serosal benzalkonium chloride (BAC) exposure. Postoperative weights were plotted. The colon segments were retrieved after 50 or 100days. Immunohistochemical staining (IHC) for beta-III tubulin (TUJ1) and glial fibrillary acid protein (GFAP) revealed colonic ganglia. Muscle layer diameter and the presence of ganglia were contrasted between normal and denervated segments. RESULTS All animals survived, experienced 5% weight loss after one week, and then consistently gained weight. Isolated segments had significantly hypertrophied smooth muscle layers compared to normal colon. Ganglia were identified by IHC in normal colonic segments, and denervated colonic segments had no IHC evidence of myenteric ganglia. CONCLUSION Colonic segmental isolation and denervation result in an effective model of irreversible colonic aganglionosis. Animals retain alimentary function. Muscularis hypertrophy, myenteric denervation, and normal animal longevity are suitable for long-term studies of cell therapy.