Stephen S. Kim

Dynamic seeding and in vitro culture of hepatocytes in a flow perfusion system.

Our laboratory has investigated hepatocyte transplantation using biodegradable polymer matrices as an alternative treatment to end-stage liver disease. One of the major limitations has been the insufficient survival of an adequate mass of transplanted cells. This study investigates a novel method of dynamic seeding and culture of hepatocytes in a flow perfusion system. In experiment I, hepatocytes were flow-seeded onto PGA scaffolds and cultured in a flow perfusion system for 24 h. Overall metabolic activity and distribution of cells were assessed by their ability to reduce MTT. DNA quantification was used to determine the number of cells attached. Culture medium was analyzed for albumin content. In Experiment II, hepatocyte/polymer constructs were cultured in a perfusion system for 2 and 7 days. The constructs were examined by SEM and histology. Culture medium was analyzed for albumin. In experiment I, an average of 4.4 X 10(6) cells attached to the scaffolds by DNA quantification. Cells maintained a high metabolic activity and secreted albumin at a rate of 13 pg/cell/day. In experiment II, SEM demonstrated successful attachment of hepatocytes on the scaffolds after 2 and 7 days. Cells appeared healthy on histology and maintained a high rate of albumin secretion through day 7. Hepatocytes can be dynamically seeded onto biodegradable polymers and survive with a high rate of albumin synthesis in the flow perfusion culture system.

Long-term follow-up of tissue-engineered intestine after anastomosis to native small bowel

BACKGROUND Our laboratory has investigated the fabrication of a tissue-engineered intestine using biodegradable polymer scaffolds. Previously we reported that isolated intestinal epithelial organoid units on biodegradable polymer scaffolds formed cysts and the neointestine was successfully anastomosed to the native small bowel. The purpose of this study was to observe the development of tissue-engineered intestine after anastomosis and to demonstrate the effect of the anastomosis over a 9-month period. METHODS Microporous biodegradable polymer tubes were created from polyglycolic acid. Intestinal epithelial organoid units were harvested from neonatal Lewis rats and seeded onto the polymers, which were implanted into the abdominal cavity of adult male Lewis rats followed by 75% small bowel resection (n=24). Three weeks after implantation, the unit/polymer constructs were anastomosed to the native jejunum in a side-to-side fashion. The anastomosed tissue-engineered intestine was measured by laparotomy 10, 24, and 36 weeks after the implantation (n= 14). During the laparotomy, all rats with an obstruction in their anastomosis were killed and excluded from the statistical analysis. Another five rats were also killed at 10 and 36 weeks for histological and morphometric studies. RESULTS All analyzed rats survived this study and significantly increased their body weight by 36 weeks. Obstruction of the anastomosis was observed in one rat at 24 weeks and in two rats at 36 weeks; however, the anastomosis was patent in the other 11 rats by 36 weeks. The tissue-engineered intestine of these 11 rats increased in length and diameter at 10, 24, and 36 weeks after anastomosis; there were statistically significant differences between each time point except between the length of 10 and 24 weeks (P<0.016 by Wilcoxon signed rank test). Histologically the inner surface of the tissue-engineered intestine was lined with well-developed neomucosa at 10 and 36 weeks; however, there were small bare areas lacking neomucosa in the tissue-engineered intestine at 36 weeks. Morphometric analysis demonstrated no significant differences in villus number, villus height, and surface length of the neomucosa at 10 and 36 weeks. CONCLUSIONS Anastomosis between tissue-engineered intestine and native small bowel resulted in no complications after operation and maintained a high patency rate for up to 36 weeks. The tissue-engineered intestine increased in size and was lined with well-developed neomucosa for the duration of the study.

Regenerative signals for intestinal epithelial organoid units transplanted on biodegradable polymer scaffolds for tissue engineering of small intestine.

BACKGROUND Our laboratory is investigating the tissue engineering of small intestine using intestinal epithelial organoid units seeded onto highly porous biodegradable polymer tubes. This study investigated methods of stimulation for optimizing neointestinal regeneration. METHODS Intestinal epithelial organoid units harvested from neonatal Lewis rats were seeded onto porous biodegradable polymer tubes and implanted into the omentum of adult Lewis rats in the following groups: (1) the control group (group C), implantation alone (n=9); (2) the small bowel resection (SBr) group, after 75% SBr (n=9); (3) the portacaval shunt (PCS) group, after PCS (n=8); and (4) the partial hepatectomy (PH) group, after 75% PH (n=8). Neointestinal cyst size was recorded using ultrasonography. Constructs were harvested at 10 weeks and were examined using histology. Morphometric analysis of the neomucosa was obtained using a computer image analysis program (NIH Image, version 1.59). RESULTS Cyst development was noted in all animals. Cyst lengths and diameters were significantly larger in the SBr group at 7 and 10 weeks compared with the other three groups (P<0.05; analysis of variance [ANOVA], Fishers protected least significant difference). Histology revealed a well-vascularized tissue with a neomucosa lining the lumen with invaginations resembling crypt-villus structures. Morphometric analysis demonstrated a significantly greater villus number, height, area, and mucosal surface in the SBr group compared with the other three groups and a significantly greater crypt number and area in the PCS group compared with group C (P<0.05; ANOVA, Fishers protected least significant difference). CONCLUSIONS Intestinal epithelial organoid units transplanted on porous biodegradable polymer tubes can successfully vascularize, survive, and regenerate into complex tissue resembling small intestine. SBr and, to a lesser extent, PCS provide significant regenerative stimuli for the morphogenesis and differentiation of tissue-engineered small intestine.

The association of cyclic parenteral nutrition and decreased incidence of cholestatic liver disease in patients with gastroschisis

PURPOSE The aim of the study was to investigate the effect of prophylactic cycling of parenteral nutrition (PN) on PN-induced cholestasis in patients with gastroschisis. METHODS Retrospective review of initial hospital admission charts for each patient with gastroschisis from 1996 to 2007 was performed. RESULTS One hundred seven patients were analyzed (36 prophylactically cycled, 71 control). Prophylactic cycling of PN was initiated at a mean age of 23 days (range, 7-89 days). Patients were followed for a total of 4255 days with 27 developing hyperbilirubinemia (cycled, 5; continuous, 22). Time to hyperbilirubinemia was longer in the prophylactically cycled group (P = .005). Cumulative incidence of hyperbilirubinemia at 25 and 50 days of PN exposure was 5.7% and 9.8% (cycled) vs 22.3% and 48.8% (continuous). At any given time, children in the continuous group were 4.76 times more likely to develop hyperbilirubinemia (95% confidence interval, 1.62-14.00). After adjusting for confounding factors, children in the continuous group were 2.86 times more likely to develop hyperbilirubinemia (95% confidence interval, 0.86-9.53), but the difference was not significant (P = .088). CONCLUSIONS Prophylactic cyclic PN is associated with a decreased incidence and prolonged time to onset of hyperbilirubinemia. Other factors, however, significantly affect this relationship. Prospective randomized investigation is warranted to investigate for a possible causal relationship.

Small intestinal submucosa as a small-caliber venous graft: A novel model for hepatocyte transplantation on synthetic biodegradable polymer scaffolds with direct access to the portal venous system☆☆☆

BACKGROUND/PURPOSE Hepatotrophic factors in the portal blood are critically important for the survival of heterotopically transplanted hepatocytes. Currently, no model exists for the implantation of hepatocytes on biodegradable polymer scaffolds with direct access to the portal blood. This study investigates the use of small intestinal submucosa (SIS) as a small-caliber venous conduit that may be used for the implantation of tissue-engineered liver. METHODS SIS was prepared from segments of rat jejunum and implanted as a venous conduit between the portal vein and inferior vena cava in 26 heparinized Lewis rats. Venograms were performed periodically, and the grafts were harvested at various time-points and examined by scanning electron microscopy (SEM) and histology. Von Willebrand Factor (vWF) staining was performed to assess endothelialization. RESULTS Five rats died of technical complications. Seventeen of 21 rats (81%) maintained patent grafts at the time of death up to 8 weeks. Venograms demonstrated patent grafts at 3 and 8 weeks. SEM results showed a smooth luminal surface with endothelial-like cells by 3 weeks. Histology demonstrated a confluent luminal endothelial monolayer, absence of thrombus, and neovascularization in the SIS graft. VWF staining results were positive, confirming the growth of endothelial cells on the luminal surface. In preliminary studies, implantation of hepatocytes seeded on biodegradable polymer tubes into the SIS graft demonstrated clusters of viable cells after 2 days. CONCLUSIONS Rat SIS can be prepared readily, maintains high patency as a small-caliber venous graft, and may be a useful model for the transplantation of tissue-engineered liver with access to the portal circulation.

The Current Status of Tissue Engineering as Potential Therapy

End-stage organ disease and tissue loss continue to be major medical problems. Although transplantation has become an established and successful method of therapy, the severe scarcity of donor organs, especially in the pediatric population, has become a major limitation and has stimulated investigation into selective cell transplantation. The authors have been investigating the fabrication of functional living tissue, or tissue engineering, using cells seeded on highly porous synthetic biodegradable polymer scaffolds as a novel approach toward the development of biological substitutes that may replace lost tissue function. Over the past decade, we have applied the principles of tissue engineering in the fabrication of a wide variety of tissues, including both structural and visceral organs. This article reviews the progress that has been achieved and the current status of tissue engineering as potential therapy for end-stage organ disease and tissue loss.

Successful anastomosis between tissue-engineered intestine and native small bowel

BACKGROUND Previous work from this laboratory has shown that isolated intestinal epithelial organoid units on porous biodegradable polymer scaffolds formed vascularized cysts lined by a neomucosa. The purpose of this study was to demonstrate anastomosis between tissue-engineered intestine and the native small bowel and to observe the effect of this anastomosis on cyst growth. METHODS Intestinal epithelial organoid units from neonatal Lewis rats were seeded onto porous biodegradable polymer tubes made of polyglycolic acid, and they were implanted into the omentum of adult male Lewis rats. Three weeks after implantation, the unit-polymer constructs were anastomosed in a side-to-side fashion to the native jejunum in 20 rats (group 1). The other 18 rats were closed without anastomosis (group 2). All 38 tissue-engineered constructs were harvested 10 weeks after implantation. Four rats underwent upper gastrointestinal (GI) study before they were killed. RESULTS The rats in group 1 increased their body weights equal to those in group 2, and there was no statistically significant difference between the two groups. Upper GI examinations revealed no evidence of either bowel stenosis or obstruction at the anastomotic site. Grossly, the patency of the anastomosis was 90% and the lumen of the cyst was visualized by the upper GI study. At the second operation, there was no significant difference in the size of the cysts in either group: however, at the time the rats were killed, the length of the cysts in group 1 was significantly longer than that in group 2 (P<0.05 using Mann-Whitney U test). Histological examination showed that cysts after anastomosis were lined by a neomucosa in continuity to native small bowel across the anastomotic site and also demonstrated crypt-villus structures. Morphometric study demonstrated that cysts in group 1 had significantly greater villus number, height, and surface length than did those in group 2. CONCLUSIONS Anastomosis between tissue-engineered intestine and native small bowel resulted in no complications after the operation, kept a high patency rate, and maintained mucosal continuity between the tissue-engineered intestine and native small bowel. Furthermore, anastomosis had a positive effect on cyst size and development of the mucosa in the tissue-engineered intestine.

Left-sided gastroschisis: higher incidence of extraintestinal congenital anomalies

PURPOSE The purpose of this study was to present 3 cases of left-sided gastroschisis and review the literature concerning this rare condition. METHODS Charts of 3 previously unreported patients with left-sided gastroschisis were reviewed. A literature review of all cases of left-sided gastroschisis was completed. RESULTS Sixteen patients with left-sided defects were identified, only 12 of whom had classic periumbilical defects. We present 3 additional patients. Eleven of 15 patients were female, with 1 patients sex not reported. Forty percent had extraintestinal anomalies. CONCLUSIONS Left-sided gastroschisis is more common in females and associated with a higher incidence of extraintestinal anomalies compared with right-sided lesions. Although the etiology remains unknown, it may differ from that of right-sided gastroschisis.

SUCCESSFUL ANASTOMOSIS BETWEEN TISSUE-ENGINEERED INTESTINE AND NATIVE SMALL BOWEL 1,2

Background. Previous work from this laboratory has shown that isolated intestinal epithelial organoid units on porous biodegradable polymer scaffolds formed vascularized cysts lined by a neomucosa. The purpose of this study was to demonstrate anastomosis between tissue-engineered intestine and the native small bowel and to observe the effect of this anastomosis on cyst growth. Methods. Intestinal epithelial organoid units from neonatal Lewis rats were seeded onto porous biodegradable polymer tubes made of polyglycolic acid, and they were implanted into the omentum of adult male Lewis rats. Three weeks after implantation, the unitpolymer constructs were anastomosed in a side-to-side fashion to the native jejunum in 20 rats (group 1). The other 18 rats were closed without anastomosis (group 2). All 38 tissue-engineered constructs were harvested 10 weeks after implantation. Four rats underwent upper gastrointestinal (GI) study before they were killed. Results. The rats in group 1 increased their body weights equal to those in group 2, and there was no statistically significant difference between the two groups. Upper GI examinations revealed no evidence of either bowel stenosis or obstruction at the anastomotic site. Grossly, the patency of the anastomosis was 90% and the lumen of the cyst was visualized by the upper GI study. At the second operation, there was no significant difference in the size of the cysts in either group: however, at the time the rats were killed, the length of the cysts in group 1 was significantly longer than that in group 2 (P<0.05 using Mann-Whitney U test). Histological examination showed that cysts after anastomosis were lined by a neomucosa in continuity to native small bowel across the anastomotic site and also demonstrated crypt-villus structures. Morphometric study demonstrated that cysts in group 1 had significantly greater villus number, height, and surface length than did those in group 2. Conclusions. Anastomosis between tissue-engineered intestine and native small bowel resulted in no complications after the operation, kept a high patency rate, and maintained mucosal continuity between the tissue-engineered intestine and native small bowel. Furthermore, anastomosis had a positive effect on cyst size and development of the mucosa in the tissue-engineered intestine. Short bowel syndrome is a clinical condition characterized by malabsorption and malnutrition after massive small bowel resection. With the development of total parenteral nutrition (TPN*), many patients may survive for an extended period of time; however, TPN is accompanied by a variety of complications, such as hepatic dysfunction, progressive renal

Thoracoscopic repair of congenital diaphragmatic hernia in infancy.

BACKGROUND Minimally invasive surgical techniques, specifically the thoracoscopic approach, have been applied to congenital diaphragmatic hernia (CDH) with varying outcomes from selected centers. The aim of our study was to examine the rate of successful completion and compare outcomes between open and thoracoscopic approaches in CDH repair. METHODS We performed a retrospective analysis of infants with CDH repair (From February 2004 to January 2008). Patients were divided into thoracoscopic and open groups, based on operative approach. We analyzed demographic, clinical, and hospitalization characteristics to compare the completion rate and outcomes in these two groups. RESULTS Analysis of 31 infants with CDH (14 thorascocopic and 17 open) demonstrated no differences in sex (P = 0.132), age (P = 0.807), birthweight (P = 0.256), weight at operation (P = 0.647), pulmonary hypertension (P = 0.067), preoperative intensive care unit (ICU) days (P = 0.673), ventilator days (P = 0.944), or use of a patch (P = 0.999) between the groups. Seventy-nine percent of thoracoscopic operative approaches were completed successfully. There was a significant difference between the open and thoracoscopic groups with respect to estimated gestational age (39 versus 36.5 weeks; P = 0.006) and operating room time (70 versus 145 minutes; P = 0.004). The total (P = 0.662), ICU (P = 0.889), and postoperative (P = 0.619) length of stay and days on ventilator (P = 0.705), as well as days until initial enteral feeds (P = 0.092), were not significantly different between groups. There were no deaths and no evidence of recurrence, with a mean follow-up of 346 days. CONCLUSIONS In our early experience, the thoracoscopic approach for congenital diaphragmatic hernia repair was completed in 80% of our patient population with minimal exclusion criteria. Further study, with larger sample sizes, is needed to ascertain differences in outcomes, such as length of stay and initiation of enteral feeding.