Regeneration of olfactory neuroepithelium in 3-methylindole-induced anosmic rats treated with intranasal chitosan.

Olfactory dysfunction significantly impairs the life quality of patients but without effective treatments to date. The previous report has demonstrated that chitosan mediates the differentiation of olfactory receptor neurons (ORNs) through insulin-like growth factors and insulin-like growth factor binding protein-2 axis in an in vitro model. However, whether chitosan can further treat olfactory dysfunction in vivo remains unexplored. This study aims to evaluate the therapeutic effect of chitosan on a 3-methylindole-induced anosmic rat model. Intraperitoneal injection of 3-methylindole is performed to induce anosmia in rats. Experimental results demonstrate that the food-finding duration after chitosan treatment gradually decrease to around 80 s, and both the olfactory neuroepithelium (ON) thickness and mature ORNs (expressing olfactory marker protein) are significantly restored. Furthermore, proliferating cells (expressing bromodeoxyuridine) are mainly co-expressed with immature ORNs (expressing ßIII tubulin) below the intermediate layer of the ON in the chitosan-treated group on day 28 following 3-methylindole treatment. Conversely, proliferating cells are scattered over the ON, and co-localized with immature ORNs and sustentacular cells (expressing keratin 18) in the sham group, and even immature ORNs go into apoptosis (expressing DNA fragmentation and cleaved caspase-3), possibly causing incomplete regeneration. Consequently, chitosan regenerates the ON by regulating olfactory neural homeostasis and reducing ORN apoptosis, and serves as a potential therapeutic intervention for olfactory dysfunction in the future.

Label-free platform on pH-responsive chitosan: Adhesive heterogeneity for cancer stem-like cell isolation from A549 cells via integrin ß4.

Great efforts have been paid to develop methodologies for cancer stem-like cell (CSLC) isolation in anti-cancer research. The major obstacle lies in the lack of generic biomarkers for different cancer types and the requirement of complicated immuno-labeling procedures. The purpose of this study is to establish a label-free platform for CSLC isolation using pH-responsive chitosan. Based on the adhesive heterogeneity, 15.7 ± 1.9 % of human non-small cell lung cancer (NSCLC) cell line A549 detached from the chitosan substrate following medium pH elevation from 6.99 to 7.65 within 1 h. As a result, this subpopulation of cells with low adhesiveness exhibited superior CSLC hallmarks, including self-renewal, invasive and metastatic potential, therapeutic-resistance, colony formation in vitro, as well as nude mice xenograft in vivo for tumorigenesis, in comparison with their high-adhesive counterpart. Furthermore, integrin ß4 is decisive in controlling CSLC detachment of NSCLC. Conclusively, this pH-dependent isolation provides new insights into biomaterial-based CSLC isolation.

Neuropeptide Y increases differentiation of human olfactory receptor neurons through the Y1 receptor.

Olfactory dysfunction significantly impedes the life quality of patients. Neuropeptide Y (NPY) is not only a neurotrophic factor in the rodent olfactory system but also an orexigenic peptide that regulates feeding behavior. NPY increases the olfactory receptor neurons (ORNs) responsivity during starvation; however, whether NPY can promote differentiation of human ORNs remains unexplored. This study investigates the effect of NPY on the differentiation of human olfactory neuroepithelial cells in vitro. Human olfactory neuroepithelium explants were cultured on tissue culture polystyrene dishes for 21 days. Then, cells were cultured with or without NPY at the concentration of 0.5 ng/mℓ for 7 days. The effects of treatment were assessed by phase contrast microscopy, immunocytochemistry and western blot analysis. The further mechanism was evaluated with NPY Y1 receptor-selected antagonist BIBP3226. NPY-treated olfactory neuroepithelial cells exhibited thin bipolar shape, low circularity, low spread area, and long processes. The expression levels of Ascl1, ßIII tubulin, GAP43 and OMP were significantly higher in NPY-treated cells than in controls (p < 0.05). NPY-treated olfactory neuroepithelial cells expressed more components of signal transduction apparatuses, Golf and ADCY3, than those without NPY treatment. Western blot analysis also further confirmed these findings (p < 0.05). Additionally, the expression levels of Ascl1, ßIII2 tubulin, GAP43, OMP, ADCY3, and Golf in BIBP3226 + NPY and controls were comparable (p > 0.05). NPY not only increases expressions of protein markers of human olfactory neuronal progenitor cells, but also promotes differentiation of ORN and enhances formation of components of olfactory-specific signal transduction pathway through Y1 receptors.

Regulation of chitosan-mediated differentiation of human olfactory receptor neurons by insulin-like growth factor binding protein-2.

Olfaction is normally taken for granted in our lives, not only assisting us to escape from dangers, but also increasing our quality of life. Although olfactory neuroepithelium (ON) can reconstitute its olfactory receptor neurons (ORNs) after injury, no adequate treatment for olfactory loss has yet emerged. The present study investigates the role of glycosaminoglycans (GAGs) in modulating olfactory neuronal homeostasis and elucidates the regulatory mechanism. This work isolates and cultures human olfactory neuroepithelial cells (HONCs) with various GAGs for 7 days, and find that chitosan promotes ORN maturation, expressing olfactory marker protein (OMP) and its functional components. Growth factor protein array, ELISA and western blot analysis reveal that insulin-like growth factor binding protein 2 (IGFBP2) shows a higher level in chitosan-treated HONCs than in controls. Biological activity of insulin-like growth factor-1 (IGF-1), IGF-2 and IGF-1 receptor (IGF1R) is further investigated. Experimental results indicate that IGF-1 and IGF-2 enhance the growth of immature ORNs, expressing ßIII tubulin, but decrease mature ORNs. Instead, down-regulation of phosphorylated IGF1R lifts the OMP expression, and lowers the ßIII tubulin expression, by incubation with the phosphorylated inhibitor of IGF1R, OSI-906. Finally, the effect of chitosan on ORN maturity is antagonized by concurrently adding IGFBP2 protease, matrix metallopeptidase-1. Overall, our data demonstrate that chitosan promotes ORN differentiation by raising the level of IGFBP2 to sequestrate the IGFs-IGF1R signaling. STATEMENT OF SIGNIFICANCE: Olfactory dysfunction serves as a crucial alarm in neurodegenerative diseases, and one of its causes is lacking of sufficient mature olfactory receptor neurons to detect odorants in the air. However, the clinical treatment for olfactory dysfunction is still controversial. Chitosan is the natural linear polysaccharide and exists in rat olfactory neuroepithelium. Previously, chitosan has been demonstrated to mediate the differentiation of olfactory receptor neurons in an in vitro rat model, but the mechanism is unknown. The study aims to evaluate the role and mechanism of chitosan in an in vitro human olfactory neurons model. Overall, these results reveal that chitosan is a potential agent for treating olfactory disorder by the maintenance of olfactory neural homeostasis. This is the first report to demonstrate that chitosan promotes differentiation of olfactory receptor neurons through increasing IGFBP2 to sequestrate the IGFs-IGF1R.

Chitosan-hyaluronan: promotion of mucociliary differentiation of respiratory epithelial cells and development of olfactory receptor neurons.

Developing a biomaterial that promotes regeneration of both respiratory epithelium (RE) and olfactory neuroepithelium (ON) improves the surgical outcome of endoscopic sinus surgery. Although chitosan (CS) inhibits mucociliary differentiation of RE, it has been reported to regenerate ON. In addition, hyaluronic acid (HA) has been demonstrated to promote regeneration of RE. Whether the composite CS + HA would simultaneously benefit RE and ON remains unexplored. Human nasal respiratory epithelial cells (RECs) and olfactory neuroepithelial cells (ONCs) are respectively obtained from the RE and the ON. They are cultured in vitro and divided into groups undergoing four treatments, control, CS, HA, and CS + HA and assessed by scanning electron microscope, immunocytochemistry, and Western blots following indicated growth conditions. RECs keep polygonal morphology with mucociliary differentiation in the CS + HA group. The levels of E-cadherin, zonula occludens-1, mucin 5AC, and forkhead box protein J1 are significantly higher in the CS + HA group than in the CS alone group. In addition, ONCs express lower cytokeratin 18 (CK18) and higher olfactory marker protein (OMP) in the CS + HA group than in HA alone group. ONCs express more signal transduction apparatuses, adenylate cyclase 3, in CS and CS + HA groups than in HA and controls. Chitosan-hyaluronan plays a part in promoting differentiation of ORNs and facilitating mucociliary differentiation of RECs. This composite is a promising biomaterial for the sinonasal application.

Hyaluronan antagonizes the differentiation effect of TGF-ß1 on nasal epithelial cells through down-regulation of TGF-ß type I receptor.

Although hyaluronan (HA)-based biomaterials have been proposed to promote mucociliary differentiation of nasal epithelial cells (NECs), the mechanism by which HA affects the growth and differentiation of NECs has not been thoroughly explored. This study investigates the effect and mechanism of HA on the differentiation of NECs. The experiment cultures human NECs in four conditions, namely controls, transforming growth factor (TGF)-ß1, TGF-ß1 + HA and HA groups. In the TGF group, the NECs become irregular shape without formation of tight junction and mucociliary differentiation of NECs is inhibited. Epithelial-mesenchymal transition (EMT) of NECs also occurs in the TGF group. However, with addition of HA in TGF groups, NECs reveal the mucociliary phenotypes of epithelial cells with tight junction expression. Incubation of TGF-ß1 in an NEC culture leads to an increase in phosphorylated type 1 TGF-ß receptors (p-TßRI). This increase is attenuated when NECs are cultured in the presence of HA. Similar expressions are observed in phosphorylated smad2/smad3. Additionally, HA-dependent inhibition of TGF-ß1 signalling is inhibited by co-incubation with a blocking antibody to CD44. Experimental results indicate that HA can antagonize TGF-ß1 effect on EMT and mucociliary differentiation of NECs by down-regulation of TßR I, which is via CD44.

Poly (ethylene-co-vinyl alcohol) is a suitable substrate for human olfactory neuroepithelial cell differentiation in vitro through a defined regulatory pathway.

Olfactory dysfunction significantly influences patients' life quality, but currently has no adequate treatment. Poly (ethylene-co-vinyl alcohol) (EVAL) mediates cell adhesion, growth and modulates differentiation of neural stem cells. However, whether EVAL is a suitable substrate to establish an in vitro culture system that can promote development and differentiation of human olfactory neuroepithelial cells (HONCs) remains unexplored. This study isolates and cultures HONCs on controls and EVAL films for 21 days. The effects of treatment are assessed using immunocytochemistry, microarray analysis, quantitative PCR, ELISA and western blots following culturing. Most of the cell morphology on controls is epithelial and expresses markers of sustentacular cells (SCs), cadherin-1 and cytokeratin18, whereas the main population on EVAL presents as morphology with extended thin processes and possesses markers of mature olfactory sensory neurons (OSNs), olfactory marker protein (OMP). Microarray analyses reveal neuropeptide Y (NPY) and amphiregulin (AREG) are the two important regulating factors on EVAL films. HONCs cultured on EVAL films enhance the development of mature OSNs through NPY signaling, and significantly decrease the growth of SCs by blocking epidermal growth factor receptor (EGFR) activation. EVAL is a potential biomaterial to serve as an ideal substrate for treating olfactory dysfunction in the future. STATEMENT OF SIGNIFICANCE: Olfaction not only contributes to enjoyments of food, but provides a clue to escape from dangerous environmental hazards. However, loss of smell is commonly progressive and there is no good prognostic approach for olfactory dysfunction. Here, we use poly (ethylene-co-vinyl alcohol) (EVAL) to establish an in vitro culture system that promotes development and differentiation of human olfactory neuroepithelial cells. We show that EVAL not only enhances the development of mature olfactory sensory neurons through neuronpeptide Y signaling, but significantly protects the olfactory neuroepithelium from metaplasia by inhibiting EGFR activation. Therefore, EVAL is a potential biomaterial to serve as an ideal substrate for treating olfactory dysfunction in the future.

Promotion of olfactory receptor neuron differentiation of olfactory neuroepithelial cells by using chitosan solution.

BACKGROUND: Olfactory dysfunction significantly influences patients' quality of life. Chitosan has been reported to support neuron and Schwann cell growth and even leads to orient axonal growth. However, researchers have yet to explore whether chitosan solution can promote differentiation of olfactory receptor neurons of the olfactory neuroepithelium and be used for treating olfactory dysfunction. OBJECTIVE: To evaluate the effect of chitosan solution on the differentiation of olfactory neuroepithelial cells. METHOD: Olfactory neuroepithelial cells were isolated from embryonic day 17 of Wistar rats and then cultured with and without soluble chitosan for 9 days. The concentration of chitosan solution was set at 0.1 mg/mL. The effects of treatment were assessed by immunocytochemistry and Western blot after culturing. RESULTS: The morphologic analysis indicated that olfactory neuroepithelial cells treated with chitosan exhibited bipolar shape with asymmetric processes. In addition, from days 3 to 9, the expression level of ßIII tubulin gradually reduced, but the expression level of olfactory marker protein significantly rose at day 9 in the chitosan groups (p < 0.05). Importantly, chitosan-treated olfactory neuroepithelial cells expressed more signal transduction apparatuses, olfactory neuron specific-G protein and adenylate cyclase 3, than those without chitosan treatment at day 9. Western blot analysis also further confirmed the results (p < 0.05). CONCLUSION: Experimental results revealed that soluble chitosan promoted differentiation of olfactory neuroepithelial cells based on its role in olfactory receptor neuron differentiation, neurite outgrowth, and signal transduction apparatus expressions.