Is nitric oxide an essential mediator in cervical inflammation and preterm birth?

OBJECTIVE: Cervical ripening is an obligatory step in the process of preterm birth. We hypothesize an inflammatory challenge to the cervix, which leads to an increase in nitric oxide production, disrupting the cervical epithelial barrier leading to preterm birth. STUDY DESIGN: For this study, three experiments were performed: (i) Using a mouse model, pregnant mice were treated with an intrauterine injection of saline or lipopolysaccharide (LPS). Mice were sacrificed and cervices were collected for molecular analysis. (ii) Immortalized ectocervical and endocervical cells were treated with either LPS or the nitric oxide donor sodium nitroprusside (SNP). Media and RNA was collected for analysis. (iii) The integrity of the epithelial cell barrier was evaluated using an in vitro permeability assay. RESULTS: The expression of inducible nitric oxide synthase (iNOS) was increased in our mouse model with LPS (p < .005). In vitro, LPS did not increase nitrate or nitrite concentrations or mRNA expression of iNOS. Permeability increased in the presence of LPS (p < .01), but was unchanged after treatment with SNP. CONCLUSIONS: These studies show that LPS increases the expression of the iNOS in an animal model of preterm birth, but the nitric oxide metabolites nitrate and nitrite do not initiate the pro-inflammatory LPS-induced breakdown of the cervical epithelial barrier.

Biomimetic and synthetic esophageal tissue engineering.

BACKGROUND/PURPOSE: A tissue-engineered esophagus offers an alternative for the treatment of pediatric patients suffering from severe esophageal malformations, caustic injury, and cancer. Additionally, adult patients suffering from carcinoma or trauma would benefit. METHODS: Donor rat esophageal tissue was physically and enzymatically digested to isolate epithelial and smooth muscle cells, which were cultured in epithelial cell medium or smooth muscle cell medium and characterized by immunofluorescence. Isolated cells were also seeded onto electrospun synthetic PLGA and PCL/PLGA scaffolds in a physiologic hollow organ bioreactor. After 2 weeks of in vitro culture, tissue-engineered constructs were orthotopically transplanted. RESULTS: Isolated cells were shown to give rise to epithelial, smooth muscle, and glial cell types. After 14 days in culture, scaffolds supported epithelial, smooth muscle and glial cell phenotypes. Transplanted constructs integrated into the host's native tissue and recipients of the engineered tissue demonstrated normal feeding habits. Characterization after 14 days of implantation revealed that all three cellular phenotypes were present in varying degrees in seeded and unseeded scaffolds. CONCLUSIONS: We demonstrate that isolated cells from native esophagus can be cultured and seeded onto electrospun scaffolds to create esophageal constructs. These constructs have potential translatable application for tissue engineering of human esophageal tissue.

Second and third trimester amniotic fluid mesenchymal stem cells can repopulate a de-cellularized lung scaffold and express lung markers.

BACKGROUND/PURPOSE: This study examined the potential of amniotic fluid mesenchymal stem cells (AF-MSCs) to generate lung precursor cells in vitro and on a xenologous three-dimensional de-cellularized lung scaffold. METHODS: AF-MSCs were isolated from human amniotic fluid obtained from 17-37 weeks gestation. Lung differentiation was induced on Matrigel or on de-cellularized rat lungs intra-tracheally injected with AF-MSCs by culturing with a modification of small airway growth medium (mSAGM) lacking retinoic acid (RA) and triodothyronine (T3) with addition of fibroblast growth factor-10 (FGF10). Cells and scaffolds were characterized by immunofluorescence and RT-PCR for markers of viability, proliferation, and lung distal airway differentiation (TTF-1(+) and SPC(+)) in the absence of markers of brain (TuJ1(-)) and thyroid (Pax8(-)). RESULTS: After culture in mSAGM on either Matrigel or lung scaffolds, there were TTF-1(+)/TuJ1(-)/Pax8(-) cells, indicating a lung precursor phenotype. In addition, SPC(+) cells also evolved suggesting a more mature lung phenotype. CONCLUSIONS: We demonstrate that mid- to late-trimester AF-MSCs can be induced to develop into lung precursor cells when cultured on the appropriate extracellular matrix (ECM), making them a viable source for use in cell therapy or development of an ex vivo tissue engineered lung.

Automated procedure for biomimetic de-cellularized lung scaffold supporting alveolar epithelial transdifferentiation.

The optimal method for creating a de-cellularized lung scaffold that is devoid of cells and cell debris, immunologically inert, and retains necessary extracellular matrix (ECM) has yet to be identified. Herein, we compare automated detergent-based de-cellularization approaches utilizing either constant pressure (CP) or constant flow (CF), to previously published protocols utilizing manual pressure (MP) to instill and rinse out the de-cellularization agents. De-cellularized lungs resulting from each method were evaluated for presence of remaining ECM proteins and immunostimulatory material such as nucleic acids and intracellular material. Our results demonstrate that the CP and MP approaches more effectively remove cellular materials but differentially retain ECM proteins. The CP method has the added benefit of being a faster, reproducible de-cellularization process. To assess the functional ability of the de-cellularized scaffolds to maintain epithelial cells, intra-tracheal inoculation with GFP expressing C10 alveolar epithelial cells (AEC) was performed. Notably, the CP de-cellularized lungs were able to support growth and spontaneous differentiation of C10-GFP cells from a type II-like phenotype to a type I-like phenotype.