Endotoxins affect diverse biological activity of chitosans in matters of hemocompatibility and cytocompatibility.

Chitosan is used in several pharmaceutical and medical applications, owing to its good cytocompatibility and hemocompatibility. However, there are conflicting reports regarding the biological activities of chitosan with some studies reporting anti-inflammatory properties while others report pro-inflammatory properties. In this regards we analyzed the endotoxin content in five different chitosans and examined these chitosans with their different deacetylation degrees for their hemocompatibility and cytocompatibility. Therefore, we incubated primary human endothelial cells or whole blood with different chitosan concentrations and studied the protein and mRNA expression of different inflammatory markers or cytokines. Our data indicate a correlation of the endotoxin content and cytokine up-regulation in whole blood for Poly-Morpho-Nuclear (PMN)-Elastase, soluble terminal complement complex SC5b-9, complement component C5/C5a, granulocyte colony-stimulating factor, Interleukin-8 (IL), IL-10, IL-13, IL-17E, Il-32α and monocyte chemotactic protein-1. In contrast, the incubation of low endotoxin containing chitosans with primary endothelial cells resulted in increased expression of E-selectin, intercellular adhesion molecule-1, vascular cell adhesion protein-1, IL-1ß, IL-6 and IL-8 in endothelial cells. We suggest that the endotoxin content in chitosan plays a major role in the biological activity of chitosan. Therefore, we strongly recommend analysis of the endotoxin concentration in chitosan, before further determining if it has pro- or anti-inflammatory properties or if it is applicable for pharmaceutical and medical fields.

Identification of an aptamer binding to human osteogenic-induced progenitor cells.

The aim of this study was to generate a specific aptamer against human jaw periosteal cells (JPCs) for tissue engineering applications in oral and maxillofacial surgery. This aptamer should serve as a capture molecule to enrich or even purify osteogenic progenitor cells from JPCs or from adult stem cells of other sources. Using systematic evolution of ligands by exponential enrichment (SELEX), we generated the first aptamer to specifically bind to human osteogenically induced JPCs. We did not detect any binding of the aptamer to undifferentiated JPCs, adipogenically and chondrogenically induced JPCs, or to any other cell line tested. However, similar binding patterns of the identified aptamer 74 were detected with mesenchymal stromal cells (MSCs) derived from placental tissue and bone marrow. After cell sorting, we analyzed the expression of osteogenic marker genes in the aptamer 74-positive and aptamer 74-negative fractions and detected no significant differences. Additionally, the analysis of the mineralization capacity revealed a slight tendency for the aptamer positive fraction to have a higher osteogenic potential. In terms of proliferation, JPCs growing in aptamer-coated wells showed increased proliferation rates compared with the controls. Herein, we report the development of an innovative approach for tissue engineering applications. Further studies should be conducted to modify and improve the specificity of the generated aptamer.

Small interfering RNA transfection against serum response factor mediates growth inhibition of benign prostatic hyperplasia fibroblasts.

To treat urethral strictures of the lower urinary tract, urethrotomy is the method of choice. But this minimally invasive method suffers from poor outcome rates and leads often to restenosis of the urinary tract because of hyper-proliferating fibroblasts. Our aim is to minimize the proliferation of excessive tissue due to a new minimal invasive therapeutic approach. As an appropriate model, we isolated fibroblasts from different benign prostatic hyperplasia patients and transfected them with small interfering RNA (siRNA) against the transcription factor serum response factor (SRF), a key factor for cell cycle regulation and apoptosis. The resulting knockdown of SRF was examined on the messenger RNA level by quantitative real-time polymerase chain reaction and on the protein level by western blot. The correlation of SRF silencing and impact on cell proliferation was examined by xCELLigence, 5-bromo-2'-deoxiuridine proliferation assay, total cell counts, and senescence assay. The transfection of primary prostatic fibroblasts with SRFsiRNA revealed specific and significant knockdown of SRF, leading to significant inhibition of proliferation after the second transfection, which was revealed by proliferation assay and total cell number. The results of this study indicate a substantial role of SRF in prostatic fibroblasts and we suggest that SRF silencing might be used for the treatment of urethral strictures to achieve a durably patent urethra.