Kim BS, Mooney DJ. Scaffolds for engineering smooth muscle under cyclic mechanical strain conditions. J Biomech Eng. 2000;122 (3) :210-5.Abstract
Cyclic mechanical strain has been demonstrated to enhance the development and function of engineered smooth muscle (SM) tissues, but appropriate scaffolds for engineering tissues under conditions of cyclic strain are currently lacking. These scaffolds must display elastic behavior, and be capable of inducing an appropriate smooth muscle cell (SMC) phenotype in response to mechanical signals. In this study, we have characterized several scaffold types commonly utilized in tissue engineering applications in order to select scaffolds that exhibit elastic properties under appropriate cyclic strain conditions. The ability of the scaffolds to promote an appropriate SMC phenotype in engineered SM tissues under cyclic strain conditions was subsequently analyzed. Poly(L-lactic acid)-bonded polyglycolide fiber-based scaffolds and type I collagen sponges exhibited partially elastic mechanical properties under cyclic strain conditions, although the synthetic polymer scaffolds demonstrated significant permanent deformation after extended times of cyclic strain application. SM tissues engineered with type I collagen sponges subjected to cyclic strain were found to contain more elastin than control tissues, and the SMCs in these tissues exhibited a contractile phenotype. In contrast, SMCs in control tissues exhibited a structure more consistent with the nondifferentiated, synthetic phenotype. These studies indicate the appropriate choice of a scaffold for engineering tissues in a mechanically dynamic environment is dependent on the time frame of the mechanical stimulation, and elastic scaffolds allow for mechanically directed control of cell phenotype in engineered tissues.
Nikolovski J, Mooney DJ. Smooth muscle cell adhesion to tissue engineering scaffolds. Biomaterials. 2000;21 (20) :2025-32.Abstract
Synthetic polyesters of lactic and glycolic acid, and the extracellular matrix molecule collagen are among the most widely-utilized scaffolding materials in tissue engineering. However, the mechanism of cell adhesion to these tissue engineering scaffolds has not been extensively studied. In this paper, the mechanism of adhesion of smooth muscle cells to these materials was investigated. Vitronectin was found to be the predominant matrix protein adsorbed from serum-containing medium onto polyglycolic acid, poly(lactic co-glycolic) acid, and collagen two-dimensional films and three-dimensional scaffolds. Fibronectin adsorbed to both materials as well, although to a much lower density. Smooth muscle cell adhesion was mediated through specific integrin receptors interacting with these adsorbed proteins, as evidenced by both immunostaining and blocking studies. The receptors involved in adhesion included the alpha(v)beta5 to vitronectin, the alpha5beta1 to fibronectin and the alpha2beta1 to collagen I. Identification of the specific receptors used to adhere to these polymers clarifies why smooth muscle tissue development differs on these scaffolds, and may allow one to design tissue formation by controlling the surface chemistry of tissue engineering scaffolds.
Kaihara S, Kim S, Kim BS, Mooney DJ, Tanaka K, Vacanti JP. Survival and function of rat hepatocytes cocultured with nonparenchymal cells or sinusoidal endothelial cells on biodegradable polymers under flow conditions. J Pediatr Surg. 2000;35 (9) :1287-90.Abstract
BACKGROUND/PURPOSE: The authors have investigated hepatocyte transplantation using biodegradable polymer scaffolds as a possible treatment of end-stage liver disease. The purpose of this study was to investigate the survival rate and function of hepatocytes alone or cocultured with other cell types on 3-dimensional biodegradable polymers for 7 days under continuous flow conditions in vitro. METHODS: Hepatocytes (group 1, n = 8), hepatocytes with nonparenchymal cells (group 2, n = 7), or hepatocytes with sinusoidal endothelial cells (group 3, n = 6) were isolated from Lewis rats and seeded onto the polymer scaffolds. The polymer devices subsequently were placed under continuous flow conditions for 7 days. Albumin production from the constructs was measured each day, and urea nitrogen synthesis was examined on day 7. The devices also were examined by histology at day 7. RESULTS: Histology results showed the presence of numerous viable hepatocytes on polymer devices, with no differences in hepatocyte viability between the 3 groups. Albumin secretion in the culture medium gradually decreased by day 7. There also were no significant differences in albumin production or urea nitrogen synthesis between the 3 groups at day 7. CONCLUSIONS: Hepatocytes could survive on the 3-dimensional polymer scaffolds under flow conditions for 7 days, and albumin secretion and urea synthesis of hepatocytes were seen at day 7. Nonparenchymal cells and sinusoidal endothelial cells had no measurable effect on hepatocyte function in our continuous flow culture system.
Bouhadir KH, Kruger GM, Lee KY, Mooney DJ. Sustained and controlled release of daunomycin from cross-linked poly(aldehyde guluronate) hydrogels. J Pharm Sci. 2000;89 (7) :910-9.Abstract
We have incorporated daunomycin, an antineoplastic agent, into a biodegradable hydrogel through a labile covalent bond. In brief, sodium alginate was chemically broken down to low molecular weight and followed by oxidation to prepare poly(aldehyde guluronate). Adipic dihydrazide was used to incorporate the drug into the polymer backbone and cross-link the polymer to form hydrogels. Daunomycin can be released from the hydrogel after the hydrolysis of the covalent linkage between the drug and the polymer. A wide range of release profiles of daunomycin (e.g., from 2 days to 6 weeks) has been achieved using these materials, and the biological activity of the released daunomycin was maintained.
Murphy WL, Peters MC, Kohn DH, Mooney DJ. Sustained release of vascular endothelial growth factor from mineralized poly(lactide-co-glycolide) scaffolds for tissue engineering. Biomaterials. 2000;21 (24) :2521-7.Abstract
Strategies to engineer bone tissue have focused on either: (1) the use of scaffolds for osteogenic cell transplantation or as conductive substrates for guided bone regeneration; or (2) release of inductive bioactive factors from these scaffold materials. This study describes an approach to add an inductive component to an osteoconductive scaffold for bone tissue engineering. We report the release of bioactive vascular endothelial growth factor (VEGF) from a mineralized, porous, degradable polymer scaffold. Three dimensional, porous scaffolds of the copolymer 85 : 15 poly(lactide-co-glycolide) were fabricated by including the growth factor into a gas foaming/particulate leaching process. The scaffold was then mineralized via incubation in a simulated body fluid. Growth of a bone-like mineral film on the inner pore surfaces of the porous scaffold is confirmed by mass increase measurements and quantification of phosphate content within scaffolds. Release of 125I-labeled VEGF was tracked over a 15 day period to determine release kinetics from the mineralized scaffolds. Sustained release from the mineralized scaffolds was achieved, and growth of the mineral film had only a minor effect on the release kinetics from the scaffolds. The VEGF released from the mineralized and non-mineralized scaffolds was over 70% active for up to 12 days following mineralization treatment, and the growth of mineral had little effect on total scaffold porosity.
Nör JE, Mitra RS, Sutorik MM, Mooney DJ, Castle VP, Polverini PJ. Thrombospondin-1 induces endothelial cell apoptosis and inhibits angiogenesis by activating the caspase death pathway. J Vasc Res. 2000;37 (3) :209-18.Abstract
Thrombospondin-1 (TSP1) is a potent natural inhibitor of angiogenesis. Although TSP1 has been reported to induce endothelial cell apoptosis in vitro and to downregulate neovascularization in vivo, the molecular mechanisms that link these two processes have yet to be established. Here we report that TSP1 mediates endothelial cell apoptosis and inhibits angiogenesis in association with increased expression of Bax, decreased expression of Bcl-2, and processing of caspase-3 into smaller proapoptotic forms. The ability of TSP1 to induce both endothelial cell apoptosis in vitro and to suppress angiogenesis in vivo was blocked by the caspase-3 inhibitor z-DEVD-FMK. TSP1 also attenuated VEGF-mediated Bcl-2 expression in endothelial cells in vitro and angiogenesis in vivo. Furthermore, TSP1 induced endothelial cell apoptosis and inhibited neovascularization in sponge implants in SCID mice. We conclude that TSP1 induces endothelial cell apoptosis and inhibits neovascularization by altering the profile of survival gene expression and activating caspase-3.
Robey TC, Eiselt PM, Murphy HS, Mooney DJ, Weatherly RA. Biodegradable external tracheal stents and their use in a rabbit tracheal reconstruction model. Laryngoscope. 2000;110 (11) :1936-42.Abstract
OBJECTIVES/HYPOTHESIS: To design and develop an external biodegradable tracheal stent for use in airway reconstructive surgery. STUDY DESIGN: Experimental model. METHODS: Biodegradable external tracheal stents were fabricated from polyglycolic acid and poly(D,L-lactide-co-glycolide) (85:15). In vitro studies were performed to analyze the mechanical properties and degradative characteristics of these stents. Then the stents were tested in vivo in an anterior tracheal reconstruction model in New Zealand white rabbits. RESULTS: The average dry modulus for the external stents was 1,800 kilopascal (kPa). All of the external stents cracked by 4 weeks in buffer solution. Significant mass loss was not appreciated until after 10 weeks in solution, but by 20 weeks the stents were nearly 100% degraded. In the in vivo portion of the study, the attrition rate for the control group was 23.1% versus 50% for the external stent group. The stridor rate was approximately 38% for both groups Of the rabbits that survived the entire 3 months of the study, the stented group, when measured by a balloon catheter method, had more patent airways than the control group, with an average stenosis of 27.8% versus 47.2%, respectively (P < .05). However, more accurate postmortem cast measurements of the internal airways did not confirm this. CONCLUSIONS: The external biodegradable tracheal stent employed in this study degraded in a predictable fashion and may provide a new method to augment surgical reconstruction of the anterior tracheal wall.
Lee KY, Peters MC, Anderson KW, Mooney DJ. Controlled growth factor release from synthetic extracellular matrices. Nature. 2000;408 (6815) :998-1000.Abstract
Polymeric matrices can be used to grow new tissues and organs, and the delivery of growth factors from these matrices is one method to regenerate tissues. A problem with engineering tissues that exist in a mechanically dynamic environment, such as bone, muscle and blood vessels, is that most drug delivery systems have been designed to operate under static conditions. We thought that polymeric matrices, which release growth factors in response to mechanical signals, might provide a new approach to guide tissue formation in mechanically stressed environments. Critical design features for this type of system include the ability to undergo repeated deformation, and a reversible binding of the protein growth factors to polymeric matrices to allow for responses to repeated stimuli. Here we report a model delivery system that can respond to mechanical signalling and upregulate the release of a growth factor to promote blood vessel formation. This approach may find a number of applications, including regeneration and engineering of new tissues and more general drug-delivery applications.
Shea LD, Wang D, Franceschi RT, Mooney DJ. Engineered bone development from a pre-osteoblast cell line on three-dimensional scaffolds. Tissue Eng. 2000;6 (6) :605-17.Abstract
Bone regeneration is based on the hypothesis that healthy progenitor cells, either recruited or delivered to an injured site, can ultimately regenerate lost or damaged tissue. Three-dimensional porous polymer scaffolds may enhance bone regeneration by creating and maintaining a space that facilitates progenitor cell migration, proliferation, and differentiation. As an initial step to test this possibility, osteogenic cells were cultured on scaffolds fabricated from biodegradable polymers, and bone development on these scaffolds was evaluated. Porous polymer scaffolds were fabricated from biodegradable polymers of lactide and glycolide. MC3T3-E1 cells were statically seeded onto the polymer scaffolds and cultured in vitro in the presence of ascorbic acid and beta-glycerol phosphate. The cells proliferated during the first 4 weeks in culture and formed a space-filling tissue. Collagen messenger RNA levels remained high in these cells throughout the time in culture, which is consistent with an observed increase in collagen deposition on the polymer scaffold. Mineralization of the deposited collagen was initially observed at 4 weeks and subsequently increased. The onset of mineralization corresponded to increased mRNA levels for two osteoblast-specific genes: osteocalcin and bone sialoprotein. Culture of cell/polymer constructs for 12 weeks led to formation of a three-dimensional tissue with architecture similar to that of native bone. These studies demonstrate that osteoblasts within a three-dimensional engineered tissue follow the classic differentiation pathway described for two-dimensional culture. Polymer scaffolds such as these may ultimately be used clinically to enhance bone regeneration by delivering or recruiting progenitor cells to the wound site.
Abraham Cohn N, Kim BS, Erkamp RQ, Mooney DJ, Emelianov SY, Skovoroda AR, O'Donnell M. High-resolution elasticity imaging for tissue engineering. IEEE Trans Ultrason Ferroelectr Freq Control. 2000;47 (4) :956-66.Abstract
An elasticity microscope provides high resolution images of tissue elasticity. With this instrument, it may be possible to monitor cell growth and tissue development in tissue engineering. To test this hypothesis, elasticity micrographs were obtained in two model systems commonly used for tissue engineering. In the first, strain images of a tissue-engineered smooth muscle sample clearly identified a several hundred micron thick cell layer from its supporting matrix. Because a one-dimensional mechanical model was appropriate for this system, strain images alone were sufficient to image the elastic properties. In contrast, a second system was investigated in which a simple one-dimensional mechanical model was inadequate. Uncultured collagen microspheres embedded in an otherwise homogeneous gel were imaged with the elasticity microscope. Strain images alone did not clearly depict the elastic properties of the hard spherical cell carriers. However, reconstructed elasticity images could differentiate the hard inclusion from the background gel. These results strongly suggest that the elasticity microscope may be a valuable tool for tissue engineering and other applications requiring the elastic properties of soft tissue at high spatial resolution (75 microm or less).
Burg KJ, Holder WD, Culberson CR, Beiler RJ, Greene KG, Loebsack AB, Roland WD, Mooney DJ, Halberstadt CR. Parameters affecting cellular adhesion to polylactide films. J Biomater Sci Polym Ed. 1999;10 (2) :147-61.Abstract
Absorbable biomaterials have been recently incorporated into the field of tissue engineering. Little work has been performed, even with the clinically acceptable absorbables, concerning their tissue promoting capability or lack, thereof. Furthermore, the relative attractions of cells to these implants may be largely disguised by the presence of serum. This research involved the development of an adhesion assay to compare the adhesion behavior of two cell types to two different polylactides in a serum free environment. The results showed that the attachment behavior depends not only on the cell or the polymer but a combination of the two.
Cahill MT, Moriarty PM, Mooney DJ, Kennedy SM. Pilomatrix carcinoma of the eyelid. Am J Ophthalmol. 1999;127 (4) :463-4.Abstract
PURPOSE: To report a case of a recurring mass with an unusual origin on the eyelid of a 34-year-old man. METHOD: Case report. RESULT: Histology demonstrated that the mass was a pilomatrix carcinoma. CONCLUSION: An atypical mass with unusual symptoms or signs needs definitive treatment and diagnostic confirmation with histology.
Baum BJ, Wang S, Cukierman E, Delporte C, Kagami H, Marmary Y, Fox PC, Mooney DJ, Yamada KM. Re-engineering the functions of a terminally differentiated epithelial cell in vivo. Ann N Y Acad Sci. 1999;875 :294-300.Abstract
Because of their easy access, and important role in oral homeostasis, mammalian salivary glands provide a unique site for addressing key issues and problems in tissue engineering. This manuscript reviews studies by us in three major directions involving re-engineering functions of salivary epithelial cells. Using adenoviral-mediated gene transfer in vivo, we show approaches to i) repair damaged, hypofunctional glands and ii) redesign secretory functions to include endocrine as well as exocrine pathways. The third series of studies show our general approach to develop an artificial salivary gland for clinical situations in which all glandular tissue has been lost.
Rowley JA, Madlambayan G, Mooney DJ. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials. 1999;20 (1) :45-53.Abstract
Alginate hydrogels are used extensively in cell encapsulation, cell transplantation, and tissue engineering applications. Alginates possess many favorable properties required in biomaterials, but are unable to specifically interact with mammalian cells. We have therefore covalently modified alginate polysaccharides with RGD-containing cell adhesion ligands utilizing aqueous carbodiimide chemistry. The chemistry has been optimized and quantified with reaction efficiencies reaching 80% or greater. The concentration of peptide available for reaction was then varied to create hydrogels with a range of ligand densities. Mouse skeletal myoblasts were cultured on alginate hydrogel surfaces coupled with GRGDY peptides to illustrate achievement of cellular interaction with the otherwise non-adhesive hydrogel substrate. Myoblasts adhere to GRGDY-modified alginate surfaces, proliferate, fuse into multinucleated myofibrils, and express heavy-chain myosin which is a differentiation marker for skeletal muscle. Myoblast adhesion and spreading on these GRGDY-modified hydrogels was inhibited with soluble ligand added to the seeding medium, illustrating the specificity of adhesion to these materials. Alginate may prove to be an ideal material with which to confer specific cellular interactive properties, potentially allowing for the control of long-term gene expression of cells within these matrices.
Kim SS, Kaihara S, Benvenuto MS, Choi RS, Kim BS, Mooney DJ, Taylor GA, Vacanti JP. Regenerative signals for intestinal epithelial organoid units transplanted on biodegradable polymer scaffolds for tissue engineering of small intestine. Transplantation. 1999;67 (2) :227-33.Abstract
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], Fisher's 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, Fisher's 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.
Kim SS, Kaihara S, Benvenuto MS, Kim BS, Mooney DJ, Vacanti JP. 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. J Pediatr Surg. 1999;34 (1) :124-8.Abstract
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.
Nör JE, Christensen J, Mooney DJ, Polverini PJ. Vascular endothelial growth factor (VEGF)-mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl-2 expression. Am J Pathol. 1999;154 (2) :375-84.Abstract
Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen and permeability factor that is potently angiogenic in vivo. We report here studies that suggest that VEGF potentiates angiogenesis in vivo and prolongs the survival of human dermal microvascular endothelial cells (HDMECs) in vitro by inducing expression of the anti-apoptotic protein Bcl-2. Growth-factor-enriched and serum-deficient cultures of HDMECs grown on collagen type I gels with VEGF exhibited a 4-fold and a 1.6-fold reduction, respectively, in the proportion of apoptotic cells. Enhanced HDMEC survival was associated with a dose-dependent increase in Bcl-2 expression and a decrease in the expression of the processed forms of the cysteine protease caspase-3. Cultures of HDMECs transduced with and overexpressing Bcl-2 and deprived of growth factors showed enhanced protection from apoptosis and exhibited a twofold increase in cell number and a fourfold increase in the number of capillary-like sprouts. HDMECs overexpressing Bcl-2 when incorporated into polylactic acid sponges and implanted into SCID mice exhibited a sustained fivefold increase in the number of microvessels and a fourfold decrease in the number of apoptotic cells when examined 7 and 14 days later. These results suggest that the angiogenic activity attributed to VEGF may be due in part to its ability to enhance endothelial cell survival by inducing expression of Bcl-2.
Kim BS, Nikolovski J, Bonadio J, Mooney DJ. Cyclic mechanical strain regulates the development of engineered smooth muscle tissue. Nat Biotechnol. 1999;17 (10) :979-83.Abstract
We show that the appropriate combinations of mechanical stimuli and polymeric scaffolds can enhance the mechanical properties of engineered tissues. The mechanical properties of tissues engineered from cells and polymer scaffolds are significantly lower than the native tissues they replace. We hypothesized that application of mechanical stimuli to engineered tissues would alter their mechanical properties. Smooth muscle tissue was engineered on two different polymeric scaffolds and subjected to cyclic mechanical strain. Short-term application of strain increased proliferation of smooth muscle cells (SMCs) and expression of collagen and elastin, but only when SMCs were adherent to specific scaffolds. Long-term application of cyclic strain upregulated elastin and collagen gene expression and led to increased organization in tissues. This resulted in more than an order of magnitude increase in the mechanical properties of the tissues.
Shea LD, Smiley E, Bonadio J, Mooney DJ. DNA delivery from polymer matrices for tissue engineering. Nat Biotechnol. 1999;17 (6) :551-4.Abstract
We have proposed engineering tissues by the incorporation and sustained release of plasmids encoding tissue-inductive proteins from polymer matrices. Matrices of poly(lactide-co-glycolide) (PLG) were loaded with plasmid, which was subsequently released over a period ranging from days to a month in vitro. Sustained delivery of plasmid DNA from matrices led to the transfection of large numbers of cells. Furthermore, in vivo delivery of a plasmid encoding platelet-derived growth factor enhanced matrix deposition and blood vessel formation in the developing tissue. This contrasts with direct injection of the plasmid, which did not significantly affect tissue formation. This method of DNA delivery may find utility in tissue engineering and gene therapy applications.
Kim SS, Kaihara S, Benvenuto MS, Choi RS, Kim BS, Mooney DJ, Vacanti JP. Effects of anastomosis of tissue-engineered neointestine to native small bowel. J Surg Res. 1999;87 (1) :6-13.Abstract
BACKGROUND: Our laboratory is investigating the tissue engineering of small intestine using intestinal epithelial organoid units seeded onto highly porous biodegradable polymer matrices. This study investigated the effects of anastomosis of tissue-engineered intestine to native small bowel alone or combined with small bowel resection on neointestinal regeneration. METHODS: Intestinal epithelial organoid units harvested from neonatal Lewis rats were seeded onto biodegradable polymer tubes and implanted into the omentum of adult Lewis rats as follows: (1) implantation alone (n = 9); (2) implantation followed by anastomosis to native small bowel at 3 weeks (n = 11); and (3) implantation after small bowel resection and anastomosis to native small bowel at 3 weeks (n = 8). All constructs were harvested at 10 weeks and examined by histology. Morphometric analysis of the neomucosa was obtained using a computer image analysis program. RESULTS: Cyst development was noted in all animals. All anastomoses were patent at 10 weeks. Histology revealed the development of a vascularized tissue with a neomucosa lining the lumen of the cyst with invaginations resembling crypt-villus structures. Morphometric analysis demonstrated significantly greater villus number, villus height, crypt number, crypt area, and mucosal surface length in groups 2 and 3 compared with group 1, and significantly greater villus number, villus height, crypt area, and mucosal surface length in group 3 compared with group 2 (P < 0.05, ANOVA, Tukey test). CONCLUSION: Intestinal epithelial organoid units transplanted on biodegradable polymer tubes can regenerate into complex tissue resembling small intestine. Anastomosis to native small bowel combined with small bowel resection and anastomosis alone contribute significant regenerative stimuli for the morphogenesis and differentiation of tissue-engineered neointestine.