Heart valve tissue engineering offers the promise of improved treatments for congenital heart disorders; however, widespread clinical availability of a tissue engineered heart valve (TEHV) has been hindered by scientific and regulatory concerns, including the lack of a disposable, bioreactor system for nondestructive valve seeding and mechanical conditioning. Here we report the design for manufacture and the production of full scale, functional prototypes of such a system. To evaluate the efficacy of this bioreactor as a tool for seeding, ovine aortic valves were decellularized and subjected to seeding with human mesenchymal stem cells (hMSC). The effects of pulsatile conditioning using cyclic waveforms tuned to various negative and positive chamber pressures were evaluated, with respect to the seeding of cells on the decellularized leaflet and the infiltration of seeded cells into the interstitium of the leaflet. Infiltration of hMSCs into the aortic valve leaflet was observed following 72 h of conditioning under negative chamber pressure. Additional conditioning under positive pressure improved cellular infiltration, while retaining gene expression within the MSC-valve interstitial cell phenotype lineage. This protocol resulted in a subsurface pilot population of cells, not full tissue recellularization. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 249-259, 2017.

In tissue engineering, an ideal scaffold attracts and supports cells thus providing them with the necessary mechanical support and architecture as they reconstruct new tissue in vitro and in vivo. This manuscript details a novel matrix derived from decellularized Wharton's jelly (WJ) obtained from human umbilical cord for use as a scaffold for tissue engineering application. This decellularized Wharton's jelly matrix (DWJM) contained 0.66 +/- 0.12 mu g/mg sulfated glycosaminoglycans (GAGs), and was abundant in hyaluronic acid, and completely devoid of cells. Mass spectroscopy revealed the presence of collagen types II, VI and XII, fibronectin- I, and lumican I. When seeded onto DWJM, WJ mesenchymal stem cells (WJMSCs), successfully attached to, and penetrated the porous matrix resulting in a slower rate of cell proliferation. Gene expression analysis of WJ and bone marrow (BM) MSCs cultured on DWJM demonstrated decreased expression of proliferation genes with no clear pattern of differentiation. When this matrix was implanted into a murine calvarial defect model with, green fluorescent protein (GFP) labeled osteocytes, the osteocytes were observed to migrate into the matrix as early as 24 hours. They were also identified in the matrix up to 14 days after transplantation. Together with these findings, we conclude that DWJM can be used as a 3D porous, bioactive and biocompatible scaffold for tissue engineering and regenerative medicine applications.

Cardiopulmonary bypass (CPB) protocols of the baboon (Papio cynocephalus anubis) are limited to obtaining experimental data without concern for long-term survival. In the evaluation of pulmonary artery tissue engineered heart valves (TEHVs), pediatric CPB methods are adapted to accommodate the animals' unique physiology enabling survival up to 6 months until elective sacrifice. Aortic access was by a 14F arterial cannula and atrial access by a single 24F venous cannula.The CPB circuit includes a 3.3 L/min flow rated oxygenator, 1/4" x %" arterial-venous loop, 3/8" raceway, and bubble trap. The prime contains 700 mL Plasma-Lyte, 700 units heparin, 5 mL of 50% dextrose, and 20 mg amiodarone. Heparinization (200 u/kg) targets an activated clotting time of 350 seconds. Normothermic CPB was initiated at a 2.5 L/m2/min cardiac index with a mean arterial pressure of 55-80 mmHg. Weaning was monitored with transesophageal echocardiogram. Post-CPB circuit blood was re-infused. Chest tubes were removed with cessation of bleeding. Extubation was performed upon spontaneous breathing. The animals were conscious and upright 3 hours post-CPB. Bioprosthetic valves or TEHVs were implanted as pulmonary replacements in 20 baboons: weight = 27.5 +/- 5.6 kg, height = 73 +/- 7 cm, body surface area = 0.77 m2 +/- 0.08, mean blood flow = 1.973 +/- .254 L/min, core temperature = 37.1 +/- .1 degree C, and CPB time = 60 +/- 40 minutes. No acidosis accompanied CPB. Sixteen animals survived, four expired. Three died of right ventricular failure and one of an anaphylactoid reaction. Surviving animals had normally functioning replacement valves and ventricles. Baboon CPB requires modifications to include high systemic blood pressure for adequate perfusion into small coronary arteries, careful CPB weaning to prevent ventricular distention, and drug and fluid interventions to abate variable venous return related to a muscularized spleno-splanchnic venous capacity.

Background. This study examines in vitro seeding of decellularized human pulmonary valves (hPVs) with human valve interstitial cells (hVICs) isolated from unrelated donor aortic valve leaflets. An assay was developed to assess seeding using precut uniform sized biopsies from whole hPVs for sequential evaluation of seeding efficiency, proliferation, and migration.Materials and Methods. Scaffolds for seeding were created from decellularized hPVs using a reciprocating osmolality, double detergent, enzyme, multiple solvent protocol. hVICs seeded decellularized leaflet and sinus wall scaffolds were incubated in either static or cyclic pressure bioreactors. Low, medium, and high initial cell seeding "dosing" densities were assayed at subsequent three time points, using eight replicates each (n=576 biopsies including manufactured scaffold controls). Metabolically viable seeded cells were quantified by MTT assay. Histology defined cell locations and morphology.Results. After 24 h of static seeding with 2.5 x 10(5) cells (medium dose), 100 +/- 13 cells/mm(2) (2.5%) attached to leaflets, compared with 193 +/- 21 cells/mm(2) (8%) for sinuses. Subsequent 4 d in static culture yielded 894 +/- 84 and 838 +/- 50 cells/mm(2) versus pulsatile culture yielding 80 +/- 12 and 79 +/- 12 cells/mm(2) for leaflet and sinus, respectively. However, 76.0% +/- 12.2% of cells in leaflets in the pulsatile bioreactor were subsurface as compared to 21.4% +/- 3.9% in statically cultured leaflets (P < 0.001).Conclusion. Different seeding modes suggest a tradeoff between surface proliferation resulting in higher absolute cell numbers for static seeding versus fewer cells in a cyclic pressure bioreactor but with a greater percentage having migrated into the matrix. The medium seeding dose determined to be optimal is actually feasible for tissue engineering heart valves, and can be achieved by fairly traditional cell amplification methods. (C) 2012 Elsevier Inc. All rights reserved.

BACKGROUND:The organogenesis of esophageal atresia with tracheoesophageal fistula remains unclear. We have previously demonstrated that the fistula tract develops from a trifurcation of the embryonic lung bud and displays pulmonary lineage traits. Unlike the lung, the fistula grows without branching. Bone morphogenetic proteins (BMPs) are known to be important in lung branching. We studied possible BMP signaling defects as a potential cause for the absence of branching in the fistula tract. METHODS:Adriamycin was administered to pregnant rats on days 6-9 of gestation to induce tracheoesophageal fistula. Microdissection was performed at E13 and E17 isolating the foregut. Tissues were analyzed using immunohistochemistry for BMP ligand (BMP2, BMP4, BMP7) and receptor (BMPRIA, BMPRIB, BMPRII) expression. RESULTS:Immunohistochemistry revealed the presence of all 3 BMP ligands at E13, localized specifically to the esophageal mucosa but absent in the fistula and lung. At E17, the ligands were again present in the esophageal mucosa, and additionally in the fistula tract mucosa, but remained absent in the lung. At E17, all of the BMP receptors were also localized to the luminal surface of esophagus and fistula. However, in the lung epithelium, only BMPRII was found, whereas BMPRIA and BMPRIB remained absent. CONCLUSIONS:The normal expression pattern of BMP4 was increased at the branch tips and low between branches. Among other results, we show here a constant expression level of BMP ligands throughout the entire epithelium of the fistula tract. This diffuse expression suggests defective BMP signaling in the fistula tract and explains its nonbranching phenotype.

The organogenesis of esophageal atresia with tracheoesophageal fistula (EA/TEF) remains unknown. The fistula tract appears to develop from a non-branching trifurcation of the embryonic lung bud. The non-branching growth of the fistula differs from the other lung buds and suggests a deficiency in bone morphogenetic protein (BMP) signaling, since BMPs are critical to proper lung development and branching. With IRB approval, portions of newborn human proximal esophageal pouch and distal fistula samples were recovered at the time of surgical repair of EA/TEF. The tissues were processed for immunohistochemistry. Commercially available fetal tissues were used as controls. In control tissues, BMP ligands (BMP 2, 4, and 7) were all present in the esophagus but absent in the trachea. BMPRIA was absent in both tissues. BMPRIB was detected in trachea but not in esophagus and BMPRII was detected in esophagus but not in trachea. In the EA/TEF specimens, all BMP ligands were present in the proximal esophageal pouch but absent in the fistula tract. BMPRIA and BMPRIB were not detected in either tissue. However, BMPRII was found in both fistula tract and proximal pouch. The submucosa of the fistula appears to maintain a mixed (identical neither to lung, esophagus, or trachea) BMP signaling pattern, providing one mechanism which could potentially explain the esophageal dismotility and lack of lung branching seen in the fistula/distal esophagus.

Epithelial–mesenchymal interactions are crucial for the proper development of many organs, including the pancreas. Within the pancreas, the ducts are thought to harbor stem/progenitor cells, and possibly to give rise to pancreatic ductal carcinoma. Little is known about the mechanism of formation of pancreatic ducts in the embryo. Pancreatic mesenchyme contains numerous soluble factors which help to sustain the growth and differentiation of exocrine and endocrine structures. Here, we report that one such morphoregulatory mesenchymal protein, epimorphin, plays an important role during pancreatic ductal proliferation and differentiation. We found that epimorphin is expressed in pancreatic mesenchyme during early stages of development, and at mesenchymal–epithelial interfaces surrounding the ducts at later stages. Strong upregulation of epimorphin expression was seen during in vitro pancreatic duct differentiation. Similarly, in vitro pancreatic duct formation was inhibited by a neutralizing antibody against epimorphin, whereas addition of recombinant epimorphin partially rescued duct formation. Together, our study demonstrates the role of epimorphin in pancreatic ductal morphogenesis.

A key goal of cellular engineering is to manipulate progenitor cells to become beta-cells, allowing cell replacement therapy to cure diabetes mellitus. As a paradigm for cell engineering, we have studied the molecular mechanisms by which AR42J cells become beta-cells. Bone morphogenetic proteins (BMPs), implicated in a myriad of developmental pathways, have not been well studied in insulin-positive differentiation. We found that the canonical intracellular mediators of BMP signaling, Smad-1 and Smad-8, were significantly elevated in AR42J cells undergoing insulin-positive differentiation in response to exendin-4 treatment, suggesting a role for BMP signaling in beta-cell formation. Similarly, endogenous BMP-2 ligand and ALK-1 receptor (activin receptor-like kinase-1; known to activate Smads 1 and 8) mRNAs were specifically up-regulated in exendin-4-treated AR42J cells. Surprisingly, Smad-1 and Smad-8 levels were suppressed by the addition of BMP-soluble receptor inhibition of BMP ligand binding to its receptor. Here, insulin-positive differentiation was also ablated. BMP-2 ligand antisense also strongly inhibited Smad-1 and Smad-8 expression, again with the abolition of insulin-positive differentiation. These results demonstrate a previously unrecognized key role for BMP signaling in mediating insulin-positive differentiation through the intracellular Smad signaling pathway. In short, BMP signaling may represent a novel downstream target of exendin-4 (glucagon-like peptide 1) signaling and potentially serve as an upstream regulator of transforming growth factor-beta isoform signaling to differentiate the acinar-like AR42J cells into insulin-secreting cells.

The differentiation of pancreatic exocrine AR42J cells into insulin-expressing endocrine cells has served as an important model for both endogenous in vivo beta-cell differentiation as well as potential application to beta-cell engineering of progenitor cells. Exogenous activin, possibly working through intracellular smad 2 and/or smad 3, as well as exogenous exendin-4 (a long-acting glucagon-like peptide-1 agonist) have both been shown to induce insulin-positive/endocrine differentiation in AR42J cells. In this study, we present evidence of significant interplay and interdependence of these two pathways as well as potential synergy between the pathways. In particular, insulin-positive differentiation seems to entail an exendin-4-induced drop in smad 2 and elevation in smad 3 in RNA levels. The latter appears to be dependent on endogenous transforming growth factor (TGF)-beta isoform release by the AR42J cells and may serve as a mechanism to promote beta-cell maturation. The drop in smad 2 may mediate early endocrine commitment. The coapplication of exogenous exendin-4 and, specifically, low-dose exogenous TGF-beta1 led to a dramatic 20-fold increase in insulin mRNA levels, supporting a novel synergistic and codependent relationship between exendin-4 signaling and TGF-beta isoform signaling.

Introduction: During embryogenesis, developing organs are in direct apposition to one another. The coelomic epithelium (peritoneal anlage) has barrier properties and its removal adversely affects development. We hypothesize that coelomic epithelium contains signals within a developing organ to prevent inappropriate attenuation of signals.

Introduction: TGF-beta isoforms are a multifunctional set of protein that regulates cell proliferation, differentiation and extracellular matrix synthesis. A few studies have suggested a role for TGF-beta in pancreatic organogenesis. We studied TGF beta signaling in the developing pancreas using ontogenic and inhibitory studies.

Abstract Introduction: TGF-beta signaling has been implicated in pancreatic cancer due to the prevalence of smad4 mutations. An established model for pancreatic endocrine differentiation is exendin-4 (glucagon-like peptide-1 analog) treatment of AR42J cells (rat pancreatic carcinoma cell line). We studied TGF-beta superfamily signaling and downstream intracellular effectors (smad proteins) implicated in many developmental processes. Here, we found a novel interdependence between GLP-1 and TGF-beta signaling essential for endocrine differentiation. Methods: AR42J cells were cultured with exendin-4 at serial doses for three days. For inhibitory studies, morpholino antisense and missense controls were added to culture media. Other bioactivity studies were performed using either TGF-beta ligands or else a pan-specific TGF-beta neutralizing antibody. Cell differentiation was analyzed by semi-quantitative PCR, as well as immunocytochemistry (IFCC). Results: Smad2 and smad3 mRNA levels were respectively decreased and increased in response to exendin-4 treatment. Further, smad2 antisense or smad2/smad3 double antisense prevented AR42J cells from becoming insulin-positive. Surprisingly, smad3 antisense alone led to a 6.5-fold enhancement of insulin mRNA levels and a doubling of insulin-positive cells by IFCC. The addition of TGF-beta1 ligand greatly upregulated smad3 mRNA and led to a dramatic elevation in insulin expression. Interestingly, TGF-beta smads and insulin-positive differentiation were completely blocked by TGF-beta antibodies, or TGF-beta receptor type I antisense (Alk-1 and Alk-5). Conclusions: TGF-Beta isoform signaling represents a key control point in regulating the level of differentiation of pancreatic AR42J cancer cells.

Introduction: The organogenesis of esophageal atresia with tracheoesophageal fistula (EA/TEF) remains unknown. The fistula tract appears to develop from a non-branching trifurcation of the embryonic lung bud. The non-branching growth of the fistula differs from the other lung buds and suggests a deficiency in Bone Morphogenetic Protein (BMP) signaling, since BMP’s are critical to proper lung development and branching.