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Articles by Tracy C. Grikscheit in JoVE

 JoVE Bioengineering

Tissue Engineering of the Intestine in a Murine Model

1Children's Hospital Los Angeles, Division of Pediatric Surgery, Saban Research Institute, Keck School of Medicine of the University of Southern California


JoVE 4279

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Other articles by Tracy C. Grikscheit on PubMed

The History and Current Status of Tissue Engineering: The Future of Pediatric Surgery

Progress in the pediatric surgical treatment of numerous tissue deficits has been achieved through the relatively new field of tissue engineering. Tissue engineering has distinct advantages over native tissue or prosthetic substitution including self-repair and growth with the patient, avoiding multiple surgeries. The application of microfabrication has allowed more precision in the control of cell interactions and resulting tissue architecture.

Tissue-engineered Colon Exhibits Function in Vivo

Postcolectomy morbidities include important changes in enterohepatic circulation, stool microbiology, and absorption. The surgical substitution of an ileal pouch for the absent colon also has a number of serious complications. We report in vivo colon replacement by tissue-engineered colon (TEC) in lieu of an ileal pouch.

Tissue-engineered Small Intestine: Ontogeny of the Immune System

Using tissue-engineering techniques, we have developed a that regenerates structural and transporter properties of native jejunum. The purpose of this study was to characterize the mucosal immune system of the engineered neointestine. We hypothesized that the neointestinal mucosa is capable of developing a mature immunocyte population and that exposure to luminal stimuli is critical to this development.

Tissue-engineered Large Intestine Resembles Native Colon with Appropriate in Vitro Physiology and Architecture

Novel production and in vitro characterization of tissue engineered colon.

Effect of GLP-2 on Mucosal Morphology and SGLT1 Expression in Tissue-engineered Neointestine

Using tissue-engineering techniques, we have developed a neointestine that regenerates the structural and dynamic features of native small intestine. In this study, we tested neointestinal responsiveness to glucagon-like peptide 2 (GLP-2). Neointestinal cysts were engineered by seeding biodegradable polymers with neonatal rat intestinal organoid units. The cysts were matured and anastomosed to the native jejunum of syngeneic adult recipients. Animals were treated with GLP-2 [Gly2] (twice daily, 1 microg/g body wt) or vehicle alone (control) for 10 days. Rats were then killed, and tissues were harvested for analysis. Na+-glucose cotransporter (SGLT1) mRNA expression was assessed with Northern blotting and in situ hybridization. SGLT1 protein was localized by using immunofluorescence. GLP-2 administration resulted in 1.8- and 1.7-fold increases (P < 0.05) in neointestinal villus height and crypt depth, respectively. GLP-2 administration also resulted in a 2.4-fold increase (P < 0.01) in neomucosal SGLT1 mRNA expression. SGLT1 mRNA expression was localized to enterocytes throughout the villi, and SGLT1 protein was localized to the brush border of enterocytes along the entire length of villi from the neointestine of GLP-2-treated animals. The response of tissue-engineered neointestine to exogenous GLP-2 includes mucosal growth and enhanced SGLT1 expression. Therefore, tissue-engineering principles may help in dissecting the regulatory mechanisms mediating complex processes in the intestinal epithelium.

Angiogenesis in Tissue-engineered Small Intestine

Tissue-engineered intestine offers promise as a potential novel therapy for short bowel syndrome. In this study we characterized the microvasculature and angiogenic growth factor profile of the engineered intestine. Twenty-three tissue-engineered small intestinal grafts were harvested from Lewis rat recipients 1 to 8 weeks after implantation. Architectural similarity to native bowel obtained from juvenile rats was assessed with hematoxylin and eosin-stained sections. Capillary density, measured after immunohistochemical staining for CD34, was expressed as number of capillaries per 1000 nuclei. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) tissue levels were measured by ELISA and normalized to total protein. Over the 8-week period cysts increased in volume (0.5 cm(3) at week 1 versus 12.6 cm(3) at week 8) and mass (1.30 +/- 0.29 versus 9.74 +/- 0.3 g; mean +/- SEM). Muscular and mucosal layers increased in thickness, but capillary density remained constant (82.95 +/- 4.81 capillaries per 1000 nuclei). The VEGF level was significantly higher in juvenile rat bowel than in engineered cyst (147.6 +/- 23.9 versus 42.3 +/- 3.4 pg/mg; p < 0.001). Tissue bFGF levels were also higher (315 +/- 65.48 versus 162.3 +/- 15.09 pg/mg; p < 0.05). The mechanism driving angiogenesis differs in engineered intestine and in normal bowel. VEGF and bFGF delivery may prove useful for bioengineering of intestine.

Tissue Engineering of the Gastrointestinal Tract for Surgical Replacement: a Nutrition Tool of the Future?

Optimal nutrition depends on the multiple complex functions performed by the gastrointestinal tract, which range from basic functions such as storage, conduit and mechanical processing to more finely regulated capabilities such as vectorial transport, immune defence and cell signalling. Surgical strategies to supply lacking gastrointestinal tract tissues have relied on either replacement by proxy (surgical substitution) or the introduction of prostheses. Tissue engineering seeks to replace missing tissues with engineered tissues that more accurately reproduce the native physiological and anatomical milieu. It is now possible to engineer several areas of the gastrointestinal tract with high fidelity, and to employ tissue-engineered bowel in replacement in animal models. These replacement models have reflected excellent anatomical and physiological recapitulation of native bowel by the tissue-engineered constructs in vivo.

Lymphangiogenesis in Tissue-engineered Small Intestine

Lymphangiogenesis, the formation of lymphatic vessels, has not been reported in engineered tissue. The purpose of this study was to characterize lymphangiogenesis in tissue-engineered small intestine.

Tissue-engineered Small Intestine Improves Recovery After Massive Small Bowel Resection

Rescue with tissue-engineered small intestine (TESI) after massive small bowel resection (MSBR).

Cost Considerations and Applicant Characteristics for the Pediatric Surgery Match

Formal training in pediatric surgery is highly competitive. The limited number of accredited positions has historically favored applicants with basic science experience, numerous publications, national presentations, and exposure to well-known pediatric surgeons. This review analyzes characteristics of successful applicants and cost associated with the Match.

Tissue-engineered Spleen Protects Against Overwhelming Pneumococcal Sepsis in a Rodent Model

Solid organs production is an ultimate goal of tissue engineering. After refining a technique for intestinal engineering, we applied it to a solid organ, the spleen. Overwhelming postsplenectomy sepsis results in death in nearly half of all cases. This risk is pronounced in children. Necrosis of autotransplanted spleen slices occurs prior to regeneration. We postulate that tissue engineering techniques might be superior.

Necrotizing Enterocolitis--bench to Bedside: Novel and Emerging Strategies

Necrotizing enterocolitis (NEC) is a devastating illness that predominantly affects premature neonates. The mortality associated with this disease has changed very little during the last two decades. Neonates with NEC fall into two categories: those who respond to medical management alone and those who require surgical treatment. The disease distribution may be focal, multifocal, or panintestinal. Surgical treatment should therefore be based on disease presentation. Recent studies have added significant insight into our understanding of the pathogenesis of NEC. Several groups have shown that upregulation of nitric oxide plays an integral role in the development of epithelial injury in NEC. As a result, some treatment strategies have been aimed at abrogating the toxic effects of nitric oxide. In addition, several investigators have reported the cytoprotective effect of epidermal growth factor, which is found in high levels in breast milk, on the intestinal epithelium. Thus, fortification of infant formula with specific growth factors could soon become a preferred strategy to accelerate intestinal maturation in the premature neonate to prevent the development of NEC. One of the most devastating complications of NEC is the development of short bowel syndrome (SBS). The current treatment of SBS involves intestinal lengthening procedures or bowel transplantation. A novel emerging method for treating SBS involves the use of tissue-engineered intestine. In laboratory animals, tissue-engineered small intestine has been shown to be successful in treating intestinal failure. This article examines recent data regarding surgical treatment options for NEC as well as emerging treatment modalities.

Preservation of Extracorporeal Tissue in Closing Gastroschisis Augments Intestinal Length

Prenatal closure of the umbilical ring in gastroschisis may result in an amorphous, nonviable appearing extracorporeal tissue that is resected during the repair. However, it is unclear whether such remnant intestine is truly nonviable.

Wnt5a Knock-out Mouse As a New Model of Anorectal Malformation

Anorectal malformations (ARM) represent a variety of congenital disorders that involve abnormal termination of the anorectum. Mutations in Shh signaling and Fgf10 produce a variety of ARM phenotypes. Wnt signaling has been shown to be crucial during gastrointestinal development. We therefore hypothesized that Wnt5a may play a role in anorectal development.

Tissue-engineered Small Intestine and Stomach Form from Autologous Tissue in a Preclinical Large Animal Model

Tissue-engineered small intestine, stomach, large intestine, esophagus, and gastroesophageal (GE) junction have been successfully formed from syngeneic cells, and employed as a rescue therapy in a small animal model. The purpose of this study is to determine if engineered intestine and stomach could be generated in an autologous, preclinical large animal model, and to identify if the tissue-engineered intestine retained features of an intact stem cell niche.

Massive Subcutaneous Emphysema, Pneumomediastinum, and Pneumopericardium in Children

Massive subcutaneous emphysema (SE), pneumomediastinum (PM), and pneumopericardium (PP) are rare conditions in the pediatric population. Air leak syndrome is a constellation of disorders that include SE, PM, PP, and pulmonary interstitial emphysema. In children, SE, PM, and PP are associated with obstructive airway disease most often in the case of asthma. Management may be conservative or involve invasive procedures that require surgical intervention. Here, we describe a case of massive SE, PM, and PP in a 10-year-old child after placement of a peripherally inserted central line and review the literature.

A Multicellular Approach Forms a Significant Amount of Tissue-engineered Small Intestine in the Mouse

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection. In this study, we transitioned the technique to a mouse model, allowing investigation of the processes involved during TESI formation through the transgenic tools available in this species. This is a necessary step toward applying the technique to human therapy. Multicellular organoid units were derived from small intestines of transgenic mice and transplanted within the abdomen on biodegradable polymers. Immunofluorescence staining was used to characterize the cellular processes during TESI formation. We demonstrate the preservation of Lgr5- and DcamKl1-positive cells, two putative intestinal stem cell populations, in proximity to their niche mesenchymal cells, the intestinal subepithelial myofibroblasts (ISEMFs), at the time of implantation. Maintenance of the relationship between ISEMF and crypt epithelium is observed during the growth of TESI. The engineered small intestine has an epithelium containing a differentiated epithelium next to an innervated muscularis. Lineage tracing demonstrates that all the essential components, including epithelium, muscularis, nerves, and some of the blood vessels, are of donor origin. This multicellular approach provides the necessary cell population to regenerate large amounts of intestinal tissue that could be used to treat short bowel syndrome.

Murine Tissue-engineered Stomach Demonstrates Epithelial Differentiation

Gastric cancer remains the second largest cause of cancer-related mortality worldwide. Postgastrectomy morbidity is considerable and quality of life is poor. Tissue-engineered stomach is a potential replacement solution to restore adequate food reservoir and gastric physiology. In this study, we performed a detailed investigation of the development of tissue-engineered stomach in a mouse model, specifically evaluating epithelial differentiation, proliferation, and the presence of putative stem cell markers.

VEGF Optimizes the Formation of Tissue-engineered Small Intestine

To determine the effect of VEGF overexpression on tissue-engineered small intestine (TESI) formation.

Availability of a Pediatric Trauma Center in a Disaster Surge Decreases Triage Time of the Pediatric Surge Population: a Population Kinetics Model

The concept of disaster surge has arisen in recent years to describe the phenomenon of severely increased demands on healthcare systems resulting from catastrophic mass casualty events (MCEs) such as natural disasters and terrorist attacks. The major challenge in dealing with a disaster surge is the efficient triage and utilization of the healthcare resources appropriate to the magnitude and character of the affected population in terms of its demographics and the types of injuries that have been sustained.

Tissue-engineered Small Intestine

Mesenchymal-specific Inhibition of Vascular Endothelial Growth Factor (VEGF) Attenuates Growth in Neonatal Mice

Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis and vasculogenesis. However, the role of VEGF in the regulation of neonatal mouse development is not completely defined. We sought to determine the effect of VEGF inhibition on the development of the neonatal mouse using a transgenic approach.

Treatment of Congenital Pulmonary Airway Malformation Induced Hydrops Fetalis Via Percutaneous Sclerotherapy

Large type II and III congenital pulmonary airway malformations (CPAMs) can cause pulmonary hypoplasia, non-immune hydrops fetalis and fetal demise. Fetal intervention is indicated if hydrops fetalis develops. In this report, we describe three cases of type II and III CPAMs complicated by hydrops and treated with percutaneous sclerotherapy by ethanolamine injection into the tumor. All 3 cases demonstrated reduction in size of the CPAM and resolution of the hydrops with subsequent delivery at term. We believe that fetal percutaneous sclerotherapy can be used as a minimally invasive palliative strategy to treat CPAM-induced hydrops fetalis. Further studies are needed to delineate the risks of this novel technique.

Tissue-engineering of the Gastrointestinal Tract

The purpose of this review is to describe recent advancements in tissue-engineering of the gastrointestinal system. For some patients, a congenital or acquired defect in the alimentary system results in digestive or nutritional deficiencies requiring intervention. Unfortunately, these treatments are associated with morbid complications. Advances in the growth of tissue-engineered esophagus, stomach, small intestine, colon and anus have been made in recent years. The progress reviewed here hopefully will someday benefit patients with gastrointestinal organ loss by providing a tissue replacement with morphology and function similar to native tissue.

Solid Pseudopapillary Tumor of the Pancreas: a Single-institution 20-year Series of Pediatric Patients

Solid pseudopapillary tumor (SPT) of the pancreas is a rare neoplasm. The objective of this study was to review our institution's experience and provide an update on current management in the pediatric population.

Giant Cystic Meconium Peritonitis Presenting in a Neonate with Classic Radiographic Eggshell Calcifications and Treated with an Elective Surgical Approach: a Case Report

Giant cystic meconium peritonitis is relatively rare. Patients often present with nonspecific physical findings such as distension and emesis. Plain abdominal films remain invaluable for identifying the characteristic calcifications seen with a meconium pseudocyst, and large eggshell calcifications are pathognomonic for the giant cystic subtype.

Fibroblast Growth Factor 10-fibroblast Growth Factor Receptor 2b Mediated Signaling is Not Required for Adult Glandular Stomach Homeostasis

The signaling pathways that are essential for gastric organogenesis have been studied in some detail; however, those that regulate the maintenance of the gastric epithelium during adult homeostasis remain unclear. In this study, we investigated the role of Fibroblast growth factor 10 (FGF10) and its main receptor, Fibroblast growth factor receptor 2b (FGFR2b), in adult glandular stomach homeostasis. We first showed that mouse adult glandular stomach expressed Fgf10, its receptors, Fgfr1b and Fgfr2b, and most of the other FGFR2b ligands (Fgf1, Fgf7, Fgf22) except for Fgf3 and Fgf20. Fgf10 expression was mesenchymal whereas FGFR1 and FGFR2 expression were mostly epithelial. Studying double transgenic mice that allow inducible overexpression of Fgf10 in adult mice, we showed that Fgf10 overexpression in normal adult glandular stomach increased epithelial proliferation, drove mucous neck cell differentiation, and reduced parietal and chief cell differentiation. Although a similar phenotype can be associated with the development of metaplasia, we found that Fgf10 overexpression for a short duration does not cause metaplasia. Finally, investigating double transgenic mice that allow the expression of a soluble form of Fgfr2b, FGF10's main receptor, which acts as a dominant negative, we found no significant changes in gastric epithelial proliferation or differentiation in the mutants. Our work provides evidence, for the first time, that the FGF10-FGFR2b signaling pathway is not required for epithelial proliferation and differentiation during adult glandular stomach homeostasis.

Human Tissue-engineered Colon Forms from Postnatal Progenitor Cells: an in Vivo Murine Model

Aim: Loss of colon reservoir function after colectomy can adversely affect patient outcomes. In previous work, human fetal intestinal cells developed epithelium without mesenchyme following implantation in mice. However, for humans, postnatal tissue would be the preferred donor source. We generated tissue-engineered colon (TEC) from postnatal human organoid units. Materials & methods: Organoid units were prepared from human colon waste specimens, loaded onto biodegradable scaffolds and implanted into immunocompromised mice. After 4 weeks, human TEC was harvested. Immunofluorescence staining confirmed human origin, identified differentiated epithelial cell types and verified the presence of supporting mesenchyme. Results: Human TEC demonstrated a simple columnar epithelium. Immunofluorescence staining demonstrated human origin and the three differentiated cell types of mature colon epithelium. Key mesenchymal components (smooth muscle, intestinal subepithelial myofibroblasts and ganglion cells) were seen. Conclusion: Colon can form from human progenitor cells on a scaffold in a mouse host. This proof-of-concept experiment is an important step in transitioning TEC to human therapy.

Intraoperative Hypercyanosis in a Patient with Pulmonary Artery Band: Case Report and Review of the Literature

A case of intraoperative cyanosis in a patient with a common atrioventricular canal palliated with a pulmonary artery (PA) band is presented. The patient's physiology was consistent with cyanosis due to inadequate pulmonary blood flow, and responded quickly to typical interventions used for a hypercyanotic episode in a patient with unrepaired Tetralogy of Fallot. Differences and similarities in the physiology of PA banding compared with Tetralogy of Fallot are presented, including a rationale for treatment options to treat hemodynamic decompensation occurring in the setting of anesthesia and surgery.

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