Development of Acellular Respiratory Mucosal Matrix Using Porcine Tracheal Mucosa

BACKGROUND@#Respiratory mucosa defects result in airway obstruction and infection, requiring subsequent functionalrecovery of the respiratory epithelium. Because site-specific extracellular matrix (ECM) facilitates restoration of organfunction by promoting cellular migration and engraftment, previous studies considered decellularized trachea an idealECM; however, incomplete cell removal from cartilage and mucosal-architecture destruction are frequently reported. Here,we developed a decellularization protocol and applied it to the respiratory mucosa of separated porcine tracheas. @*METHODS@#The trachea was divided into groups according to decellularization protocol: native mucosa, freezing–thawing (FT), FT followed by the use of Perasafe-based chemical agents before mucosal separation (wFTP), after mucosalseparation (mFTP), and followed by DNase decellularization (mFTD). Decellularization efficacy was evaluated by DNAquantification and hematoxylin and eosin staining, and ECM content of the scaffold was evaluated by histologic analysisand glycosaminoglycan and collagen assays. Biocompatibility was assessed by cell-viability assay and in vivotransplantation. @*RESULTS@#The mFTP mucosa showed low antigenicity and maintained the ECM to form a proper microstructure.Additionally, tonsil-derived stem cells remained viable when cultured with or seeded onto mFTP mucosa, and the in vivohost response showed a constructive pattern following implantation of the mFTP scaffolds. @*CONCLUSION@#These results demonstrated that xenogenic acellular respiratory mucosa matrix displayed suitable biocompatibilityas a scaffold material for respiratory mucosa engineering.

Development of Acellular Respiratory Mucosal Matrix Using Porcine Tracheal Mucosa

BACKGROUND@#Respiratory mucosa defects result in airway obstruction and infection, requiring subsequent functionalrecovery of the respiratory epithelium. Because site-specific extracellular matrix (ECM) facilitates restoration of organfunction by promoting cellular migration and engraftment, previous studies considered decellularized trachea an idealECM; however, incomplete cell removal from cartilage and mucosal-architecture destruction are frequently reported. Here,we developed a decellularization protocol and applied it to the respiratory mucosa of separated porcine tracheas. @*METHODS@#The trachea was divided into groups according to decellularization protocol: native mucosa, freezing–thawing (FT), FT followed by the use of Perasafe-based chemical agents before mucosal separation (wFTP), after mucosalseparation (mFTP), and followed by DNase decellularization (mFTD). Decellularization efficacy was evaluated by DNAquantification and hematoxylin and eosin staining, and ECM content of the scaffold was evaluated by histologic analysisand glycosaminoglycan and collagen assays. Biocompatibility was assessed by cell-viability assay and in vivotransplantation. @*RESULTS@#The mFTP mucosa showed low antigenicity and maintained the ECM to form a proper microstructure.Additionally, tonsil-derived stem cells remained viable when cultured with or seeded onto mFTP mucosa, and the in vivohost response showed a constructive pattern following implantation of the mFTP scaffolds. @*CONCLUSION@#These results demonstrated that xenogenic acellular respiratory mucosa matrix displayed suitable biocompatibilityas a scaffold material for respiratory mucosa engineering.

Optimization of Microenvironments Inducing Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Endothelial Cell-Like Cells

BACKGROUND: Stem cell engineering is appealing consideration for regenerating damaged endothelial cells (ECs) because stem cells can differentiate into EC-like cells. In this study, we demonstrate that tonsil-derived mesenchymal stem cells (TMSCs) can differentiate into EC-like cells under optimal physiochemical microenvironments.METHODS: TMSCs were preconditioned with Dulbecco's Modified Eagle Medium (DMEM) or EC growth medium (EGM) for 4 days and then replating them on Matrigel to observe the formation of a capillary-like network under light microscope. Microarray, quantitative real time polymerase chain reaction, Western blotting and immunofluorescence analyses were used to evaluate the expression of gene and protein of EC-related markers.RESULTS: Preconditioning TMSCs in EGM for 4 days and then replating them on Matrigel induced the formation of a capillary-like network in 3 h, but TMSCs preconditioned with DMEM did not form such a network. Genome analyses confirmed that EGM preconditioning significantly affected the expression of genes related to angiogenesis, blood vessel morphogenesis and development, and vascular development. Western blot analyses revealed that EGM preconditioning with gelatin coating induced the expression of endothelial nitric oxide synthase (eNOS), a mature EC-specific marker, as well as phosphorylated Akt at serine 473, a signaling molecule related to eNOS activation. Gelatin-coating during EGM preconditioning further enhanced the stability of the capillary-like network, and also resulted in the network more closely resembled to those observed in human umbilical vein endothelial cells.CONCLUSION: This study suggests that under specific conditions, i.e., EGM preconditioning with gelatin coating for 4 days followed by Matrigel, TMSCs could be a source of generating endothelial cells for treating vascular dysfunction.

Expression of Tenocyte Lineage-Related Factors from Tonsil-Derived Mesenchymal Stem Cells

Human palatine tonsil-derived mesenchymal stem cells (TMSCs) are known to be a new source of progenitor cells. Using waste tissue after tonsillectomy as a cell provider can be the biggest benefit of TMSCs, compared with other stem cells. The purpose of this study was to investigate tenogenic differentiation of TMSCs and to access the differential effects of transforming growth factor beta 3 (TGF-β3) on the tenogenesis of TMSCs. Human tonsil was obtained after tonsillectomy. Using a cytometric analysis, we were able to find that the TMSCs had typical mesenchymal stem cell markers: positive for CD73, CD90, and CD105, and negative for CD14, CD34, and CD45. Using TGF-β3, the expressions of tenocyte-specific genes and proteins, such as collagen type 1 (COL1), tenomodulin (TNMD), and scleraxis (SCX), were measured by a quantitative polymerase chain reaction (PCR), immunofluorescence staining, immunohistochemistry and Western blot analyses. Quantitative PCR assay showed that TGF-β3 significantly increased the expressions of tenocyte lineage marker genes, including COL1, TNMD, and SCX, at a 3-day treatment, compared with control. However, these increases were not found at long-term exposures (7 or 10 days), except that TNMD expression was maintained at 50 ng/mL at a 7-day exposure to TGF-β3. Like genes, the protein expression levels of COL1, TNMD, and SCX were also induced in TGF-β3-treated TMSCs in a 3-day treatment, which were maintained for 10 days, as evidenced by immunofluorescence staining, immunohistochemistry and Western blot analyses. This study demonstrated that TMSCs in tenogenic stimulation with TGF-β3 have a high tenogenic differentiation potential.