Human Bronchial Epithelial Cell Growth on Homologous Versus Heterologous Tissue Extracellular Matrix.

BACKGROUND: Extracellular matrix (ECM) bioscaffolds produced by decellularization of source tissue have been effectively used for numerous clinical applications. However, decellularized tracheal constructs have been unsuccessful due to the immediate requirement of a functional airway epithelium on surgical implantation. ECM can be solubilized to form hydrogels that have been shown to support growth of many different cell types. The purpose of the present study is to compare the ability of airway epithelial cells to attach, form a confluent monolayer, and differentiate on homologous (trachea) and heterologous (urinary bladder) ECM substrates for potential application in full tracheal replacement. MATERIALS AND METHODS: Porcine tracheas and urinary bladders were decellularized. Human bronchial epithelial cells (HBECs) were cultured under differentiation conditions on acellular tracheal ECM and urinary bladder matrix (UBM) bioscaffolds and hydrogels and were assessed by histology and immunolabeling for markers of ciliation, goblet cell formation, and basement membrane deposition. RESULTS: Both trachea and urinary bladder tissues were successfully decellularized. HBEC formed a confluent layer on both trachea and UBM scaffolds and on hydrogels created from these bioscaffolds. Cells grown on tracheal and UBM hydrogels, but not on bioscaffolds, showed positive-acetylated tubulin staining and the presence of mucus-producing goblet cells. Collagen IV immunolabeling showed basement membrane deposition by these cells on the surface of the hydrogels. CONCLUSIONS: ECM hydrogels supported growth and differentiation of HBEC better than decellularized ECM bioscaffolds and show potential utility as substrates for promotion of a mature respiratory epithelium for regenerative medicine applications in the trachea.

Maintenance of hepatic sinusoidal endothelial cell phenotype in vitro using organ-specific extracellular matrix scaffolds.

Sinusoidal endothelial cells (SECs) are notoriously difficult to culture in vitro. SECs represent a highly specialized endothelial cell (EC) population, and traditional methods of SEC isolation from the liver initiate a process of SEC dedifferentiation. Acellular extracellular matrix (ECM) scaffolds were investigated in a physiologically relevant in vitro culture model for their ability to maintain SEC phenotype. The cell culture model used SECs only or a coculture of SECs with hepatocytes on ECM substrates derived from the liver (L-ECM), bladder (UBM-ECM), or small intestine submucosa (SIS-ECM). The effect of the ECM substrate upon SEC dedifferentiation was evaluated using scanning electron microscopy (SEM) and confocal microscopy. When SECs alone were cultured on uncoated glass slides, collagen I, UBM-ECM, or SIS-ECM, SECs showed signs of dedifferentiation after 1 day. In contrast, SECs alone cultured on L-ECM maintained their differentiated phenotype for at least 3 days, indicated by the presence of many fenestrations on SEC surface, expression of anti-rat hepatic sinusoidal endothelial cells mouse IgG MoAb (SE-1), and lack of expression of CD31. When SECs were cocultured with hepatocytes on any of the ECM scaffolds, the SECs maintained a near-normal fenestrated phenotype for at least 1 day. However, SEM revealed that the shape, size, frequency, and organization of the fenestrations varied greatly depending on ECM source. At all time points, SECs cocultured with hepatocytes on L-ECM maintained the greatest degree of differentiation. The present study demonstrated that the acellular ECM scaffold derived from the liver maintained SEC differentiation in culture longer than any of the tested substrate materials. The replacement of complex tissues and 3-dimensional organs may require specialized scaffolds to support multiple, functional cell phenotypes.

Regenerative Medicine: lessons from Mother Nature.

Regenerative medicine strategies for the restoration of functional tissue have evolved from the concept of ex vivo creation of engineered tissue toward the broader concept of in vivo induction of functional tissue reconstruction. Multidisciplinary approaches are being investigated to achieve this goal using evolutionarily conserved principles of stem cell biology, developmental biology and immunology, current methods of engineering and medicine. This evolution from ex vivo tissue engineering to the manipulation of fundamental in vivo tenets of development and regeneration has the potential to capitalize upon the incredibly complex and only partially understood ability of cells to adapt, proliferate, self-organize and differentiate into functional tissue.

Abdominal adiposity correlates with adenotonsillectomy outcome in obese adolescents with severe obstructive sleep apnea.

Background. Obese adolescents with Obstructive Sleep Apnea (OSA) have a unique pathophysiology that combines adenotonsillar hypertrophy and increased visceral fat distribution. We hypothesized that in this population waist circumference (WC), as a clinical marker of abdominal fat distribution, correlates with the likelihood of response to AT. Methods. We conducted a retrospective cohort study of obese adolescents (BMI ≥ 97th percentile) that underwent AT for therapy of severe OSA (n = 21). We contrasted WC and covariates in a group of subjects that had complete resolution of severe OSA after AT (n = 7) with those obtained in subjects with residual OSA after AT (n = 14). Multivariate linear and logistic models were built to control possible confounders. Results. WC correlated negatively with a positive AT response in young adolescents and the percentage of improvement in obstructive apnea-hypopnea index (OAHI) after AT (P ≤ 0.01). Extended multivariate analysis demonstrated that the link between WC and AT response was independent of demographic variables, OSA severity, clinical upper airway assessment, obesity severity (BMI), and neck circumference (NC). Conclusion. The results suggest that in obese adolescents, abdominal fat distribution determined by WC may be a useful clinical predictor for residual OSA after AT.

Macrophage phenotype as a determinant of biologic scaffold remodeling.

Macrophage phenotype can be characterized as proinflammatory (M1) or immunomodulatory and tissue remodeling (M2). The present study used a rat model to determine the macrophage phenotype at the site of implantation of two biologic scaffolds that were derived from porcine small intestinal submucosa (SIS) and that differed mainly according to their method of processing: the Restore device (SIS) and the CuffPatch device (carbodiimide crosslinked form of porcine-derived SIS (CDI-SIS)). An autologous tissue graft was used as a control implant. Immunohistologic methods were used to identify macrophage surface markers CD68 (pan macrophages), CD80 and CCR7 (M1 profile), and CD163 (M2 profile) during the remodeling process. All graft sites were characterized by the dense population of CD68+ mononuclear cells present during the first 4 weeks. The SIS device elicited a predominantly CD163+ response (M2 profile, p < 0.001) and showed constructive remodeling at 16 weeks. The CDI-SIS device showed a predominately CD80+ and CCR7+ response (M1 profile, p < 0.03), and at 16 weeks was characterized by chronic inflammation. The autologous tissue graft showed a predominately CD163+ response (M2) at 1 week, with a dual M1/M2 population (CD80+, CCR7+, and CD163+) by 2 and 4 weeks and moderately well organized connective tissue by 16 weeks. The processing methods used during the manufacturing of a biologic scaffold can have a profound influence upon the macrophage phenotype profile and downstream remodeling events. Routine histologic examination alone is inadequate to determine the phenotype of mononuclear cells that participate in the host response to the scaffold.