Erin R. Ochoa

Silicon micromachining to tissue engineer branched vascular channels for liver fabrication

To date, many approaches to engineering new tissue have emerged and they have all relied on vascularization from the host to provide permanent engraftment and mass transfer of oxygen and nutrients. Although this approach has been useful in many tissues, it has not been as successful in thick, complex tissues, particularly those comprising the large vital organs such as the liver, kidney, and heart. In this study, we report preliminary results using micromachining technologies on silicon and Pyrex surfaces to generate complete vascular systems that may be integrated with engineered tissue before implantation. Using standard photolithography techniques, trench patterns reminiscent of branched architecture of vascular and capillary networks were etched onto silicon and Pyrex surfaces to serve as templates. Hepatocytes and endothelial cells were cultured and subsequently lifted as single-cell monolayers from these two-dimensional molds. Both cell types were viable and proliferative on these surfaces. In addition, hepatocytes maintained albumin production. The lifted monolayers were then folded into compact three-dimensional tissues. Thus, with the use microfabrication technology in tissue engineering, it now seems feasible to consider lifting endothelial cells as branched vascular networks from two-dimensional templates that may ultimately be combined with layers of parenchymal tissue, such as hepatocytes, to form three-dimensional conformations of living vascularized tissue for implantation.

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

Objective:Rescue with tissue-engineered small intestine (TESI) after massive small bowel resection (MSBR). Summary Background Data:Short bowel syndrome is a morbid product of massive small bowel resection. We report the first replacement of a vital organ by tissue engineering with TESI after MSBR. Methods:Ten male Lewis rats underwent TESI implantation with green fluorescent protein (GFP)-marked cells (TESI+, n = 5) or sham laparotomy (TESI−, n = 5) followed by MSBR. Side-to-side anastomosis of TESI to proximal small intestine was performed or omitted. TESIØ animals underwent implantation of engineered intestine with no further surgery. Weights were measured QOD until day 40. Transit times were measured. DNA assay was performed with computer morphometry. Northern blots of RNA were probed for intestinal alkaline phosphatase (IAP) and villin. Hematoxylin and eosin, S100, and smooth muscle actin immunohistochemistry were performed. Blood was collected at sacrifice. Results:All 10 rats initially lost then regained weight. The initial rate of weight loss was higher in TESI+ versus TESI−, but the nadir was reached a week earlier with more rapid weight gain subsequently to 98% preoperative weight on day 40 in animals with engineered intestine versus 76% (P < 0.03). Serum B12 was higher at 439 pg/mL versus 195.4 pg/mL. IAP mRNA appeared greater in TESI+ than TESIØ, with constant villin levels. Histology revealed appropriate architecture including nerve. GFP labeling persisted. Conclusions:Anastomosis of TESI significantly improved postoperative weight and B12 absorption after MSBR. IAP, a marker of differentiation in intestinal epithelium, is present in TESI, and GFP labeling was accomplished.

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

BACKGROUND 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. METHODS A short segment of jejunum or stomach was resected from 6-wk-old Yorkshire swine. Organoid units, multicellular clusters with predominantly epithelial content, were generated and loaded onto biodegradable scaffold tubes. The constructs were then implanted intraperitoneally in the autologous host. Seven wk later, all implants were harvested and analyzed using histology and immunohistochemistry techniques. RESULTS Autologous engineered small intestine and stomach formed. Tissue-engineered intestinal architecture replicated that of native intestine. Histology revealed tissue-engineered small intestinal mucosa composed of a columnar epithelium with all differentiated intestinal cell types adjacent to an innervated muscularis mucosae. Intestinal subepithelial myofibroblasts, specialized cells that participate in the stem cell niche formation, were identified. Moreover, cells positive for the putative intestinal stem cell marker, doublecortin and CaM kinase-like-1 (DCAMKL-1) expression were identified at the base of the crypts. Finally, tissue-engineered stomach also formed with antral-type mucosa (mucus cells and surface foveolar cells) and a muscularis. CONCLUSION We successfully generated tissue-engineered intestine with correct architecture, including features of an intact stem cell niche, in the pig model. To our knowledge, this is the first demonstration in which tissue-engineered intestine was successfully generated in an autologous manner in an animal model, which may better emulate a human host and the intended therapeutic pathway for humans.

Tissue-engineered esophagus: experimental substitution by onlay patch or interposition

OBJECTIVES We proposed to fabricate a tissue-engineered esophagus and to use it for replacement of the abdominal esophagus. METHODS Esophagus organoid units, mesenchymal cores surrounded by epithelial cells, were isolated from neonatal or adult rats and paratopically transplanted on biodegradable polymer tubes, which were implanted in syngeneic hosts. Four weeks later, the tissue-engineered esophagus was either harvested or anastomosed as an onlay patch or total interposition graft. Green Fluorescent Protein labeling by means of viral infection of the organoid units was performed before implantation. Histology and immunohistochemical detection of the antigen alpha-actin smooth muscle were performed. RESULTS Tissue-engineered esophagus grows in sufficient quantity for interposition grafting. Histology reveals a complete esophageal wall, including mucosa, submucosa, and muscularis propria, which was confirmed by means of immunohistochemical staining for alpha-actin smooth muscle. Tissue-engineered esophagus architecture was maintained after interposition or use as a patch, and animals gained weight on a normal diet. Green Fluorescent Protein-labeled tissue-engineered esophagus preserved its fluorescent label, proving the donor origin of the tissue-engineered esophagus. CONCLUSIONS Tissue-engineered esophagus resembles the native esophagus and maintains normal histology in anastomosis, with implications for therapy of long-segment esophageal tissue loss caused by congenital absence, surgical excision, or trauma.

Tissue-engineered large intestine resembles native colon with appropriate in vitro physiology and architecture

Objective Novel production and in vitro characterization of tissue engineered colon. Summary Background Data The colon provides important functions of short chain fatty acid production, sodium and water absorption, and storage. We report the first instance of tissue-engineered colon (TEC) production from autologous cells and its in vitro characterization. Methods Organoid units, mesenchymal cell cores surrounded by a polarized epithelia derived from full thickness sigmoid colon dissection from neonatal Lewis rats, adult rats, and tissue engineered colon itself, were implanted on a polymer scaffold into the omentum of syngeneic hosts. TEC was either anastomosed at 4 weeks or excised for Üssing chamber studies or histology, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling assay. Results TEC was generated by 100% of all animals without regard to tissue source, the first instance of engineered intestine from adult cells or an engineered tissue. TEC architecture is identical to native with muscularis propria staining for actin, acetylcholinesterase detected in a linear distribution in the lamina propria, S100-positive cells, ganglion cells, and a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling assay similar to native colon. Üssing chamber data indicated in vitro function consistent with mature colonocytes, and a positive short circuit current response to theophylline indicating intact ion transfer. TEM showed normal microarchitecture. Colon architecture was maintained in anastomosis with gross visualization of fluid uptake. Conclusions TEC can be successfully produced with fidelity to native architecture and in vitro function from neonatal syngeneic tissue, adult tissue, and TEC itself.

Dynamic rotational seeding and cell culture system for vascular tube formation.

Optimization of cell seeding and culturing is an important step for the successful tissue engineering of vascular conduits. We evaluated the effectiveness of using a hybridization oven for rotational seeding and culturing of ovine vascular myofibroblasts onto biodegradable polymer scaffolds suitable for replacement of small- and large-diameter blood vessels. Large tubes (12 mm internal diameter and 60 mm length, n = 4) and small tubes (5 mm internal diameter and 20 mm length, n = 4) were made from a combination of polyglycolic acid/poly-4-hydroxybutyrate and coated with collagen solution. Tubes were then placed in culture vessels containing a vascular myofibroblast suspension (10(6) cells/cm(2)) and rotated at 5 rpm in a hybridization oven at 37 degrees C. Light and scanning electron microscopy analyses were performed after 5, 7, and 10 days. Myofibroblasts had formed confluent layers over the outer and inner surfaces of both large and small tubular scaffolds by day 5. Cells had aligned in the direction of flow by day 7. Multiple spindle-shaped cells were observed infiltrating the polymer mesh. Cell density increased between day 5 and day 10. All conduits maintained their tubular shape throughout the experiment. We conclude that dynamic rotational seeding and culturing in a hybridization oven is an easy, effective, and reliable method to deliver and culture vascular myofibroblasts onto tubular polymer scaffolds.

An Overview of the Pathology and Approaches to Tissue Engineering

In tissue engineering, there is an attempt to culture living tissues for surgical transplantation. In vitro and in vivo approaches have produced vascular and cardiovascular components, cartilage, bone, intestine, and liver. Attempts to microdesign cell‐culture support scaffolds have used a new generation of biocompatible and bioabsorbable polymers. Suspensions of donor cells are seeded onto protein‐coated polymer scaffolds and grown to confluence in dynamic bioreactors. In vitro techniques produce monolayers of tissues. Denser masses are achieved by implanting monolayers onto a host, or by culturing cell/polymer constructs in vivo. Existing techniques have produced functioning heart valves from sheep endothelial cells and myofibroblasts. Cultured ovine arterial cells have replaced 2‐cm segments of pulmonary artery in lambs. Chondrocyte cultures have produced a human‐ear‐shaped construct, temporo‐mandibular joint discs, meniscal replacement devices, and human‐phalange‐shaped constructs, complete with a joint. The culture of composite tissue types has recently been reported. Intestinal organoid units containing a mesenchymal core with surrounding polarized epithelia have been used in lieu of an ileal pouch in Lewis rats, and the long‐term culture of rat hepatocytes has revealed cellular differentiation and neomorphology resembling elements of a biliary drainage system. To sustain the in vitro culture of dense tissues prior to implantation, micro‐electro‐mechanical systems (MEMS) fabrication technologies have been adapted to create wafers of polymer containing sealed, branching, vascular‐type spaces. After seeding with rat lung endothelial cells, followed by 5 days of bioreactor culture, the result is an endothelial network with controlled blood flow rates, pressure, and hematocrit. When these customized vascular systems can be used to support in vitro culture, a new generation of dense, composite, morphologically complex tissues will be available for clinical development.

In vitro engineering of bone using a rotational oxygen-permeable bioreactor system

Abstract Tissue engineering of bone may supersede the need in the future of autograft procedures to treat bone defects resulting from trauma or developmental diseases. A Rotational Oxygen-Permeable Bioreactor System (ROBS) has recently been developed in our laboratory to reproduce dynamic and gas-permeable culture conditions that would supply optimal oxygen and continuous loading to cell/polymer constructs in culture. The cell culture media in ROBS were examined at 1, 24 and 48 h to evaluate the kinetics of p O 2 , p CO 2 and pH without culturing cells. The results were compared to the kinetics in 100 mm diameter cell culture dishes (Control I: static, gas permeable) and 50 ml centrifuge tubes (Control II: dynamic, non-gas permeable). The results showed the same kinetics in ROBS and Control I, whereas Control II failed to maintain the gas conditions of the media. Next, osteoblasts derived from mesenchymal stromal cells (MSCs) of neonatal rats were cultured in three-dimensional poly( dl -lactide-co-glycolide) (PLGA) foams using ROBS to study the effectiveness of this bioreactor system to support cell growth and differentiation. Mineralization was observed within 2 weeks of culture and was shown throughout the polymer at 7 weeks with embedded osteocytic cells. This study demonstrates the usefulness of ROBS for in vitro bone tissue engineering.

Marginal zone B-cell lymphoma of the salivary gland arising in chronic sclerosing sialadenitis (Küttner tumor)

We report a case of extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) type of the salivary gland arising in a background of chronic sclerosing sialadenitis. Chronic sclerosing sialadenitis is a common fibrosing chronic inflammatory lesion of the submandibular gland, which is thought to be the result of sialolithiasis, and is not associated with a systemic autoimmune disease. Salivary MALT lymphomas are typically associated with lymphoepithelial sialadenitis (LESA) in a patient with or without Sjögrens syndrome. Our case of salivary MALT lymphoma was neither preceded by Sjögrens syndrome nor accompanied by LESA. This case suggests that chronic inflammatory processes other than Sjögrens syndrome may provide a substrate for the development of MALT lymphoma.

Expression of specific hepatocyte and cholangiocyte transcription factors in human liver disease and embryonic development

Transcription factors are major determinants of cell-specific gene expression in all cell types. Studies in rodent liver have shown that alterations in transcription factor expression determine lineage specification during fetal liver development and signify transdifferentiation of cells of the biliary compartment into ‘oval’ cells and eventually hepatocytes in adult liver. We examined the cellular localization of hepatocyte- or BEC-associated transcription factors in human fetal and adult liver and in diseases in which transdifferentiation between hepatocytes and biliary cells may play a role. In the normal adult human liver, hepatocyte nuclear factor (HNF)4α and HNF6 appeared exclusively in hepatocytes; HNF1β, HNF3α, and HNF3β were observed only in BEC. During fetal development both BEC and hepatocytes expressed HNF3α, HNF3β, and HNF6. HNF1α was expressed only in fetal hepatocytes. We further examined expression of transcription factors in massive hepatic necrosis and in specific types of chronic liver disease. Hepatocyte-associated transcription factors HNF4α and HNF6 also appeared in BEC in massive hepatic necrosis and chronic hepatitis C virus infection. Similarly, HNF3β that is expressed only in BEC in normal adult liver was also observed in hepatocytes in primary biliary cirrhosis and chronic biliary obstruction. These data mimic previous findings in rodents in which hepatocyte-associated transcription factors appear in biliary cells prior to emergence of oval cells, which function as progenitor cells for hepatocytes when the regenerative capacity of the latter is compromised.