Tsung-Wei Huang

Culture of nasal epithelial cells using chitosan-based membranes

The aim of this study was to evaluate whether chitosan‐based membranes can be used as scaffolds for growth and differentiation of nasal epithelial cells (NECs). Our final goal was to establish a novel methodology for enhancing the regeneration of the respiratory system.

Regulation of ciliary differentiation of human respiratory epithelial cells by the receptor for hyaluronan-mediated motility on hyaluronan-based biomaterials

Selecting a scaffold that facilitates ciliary differentiation of respiratory epithelial cells (RECs) is crucial in developing tissue engineered respiratory epithelium. Hyaluronan derivative membranes, consisting of an esterified form of hyaluronan (HYAFF), have been proved to promote ciliary differentiation of RECs in the presence of retinoic acid (RA). However, the regulatory mechanism of ciliary differentiation-promoting effect of hyaluronan-based biomaterials remains unknown. In addition to investigating the ciliary differentiation of RECs on HYAFF with/without RA compared with that on collagen with/without RA, this study elucidates the role of the receptor for hyaluronan-mediated motility (RHAMM) in promoting ciliary differentiation of RECs. Analytical results of culturing RECs on collagen and HYAFF indicate that only HYAFF can increase the ciliary differentiation of RECs under RA-free conditions. The expression level of RHAMM mRNA of RECs more significantly decreases on collagen than that on HYAFF without RA. Therefore, by using lentiviral vector-based short hairpin RNA targeting RHAMM, the study further reveals that knockdown of RHAMM obviously inhibits the ciliary differentiation of RECs on collagen with RA and on HYAFF with/without RA. In addition to demonstrating that hyaluronan-based biomaterials partially replace RA in the ciliary differentiation of RECs, which is regulated by RHAMM, this study establishes that RHAMM regulates the ciliary differentiation-promoting effect of RA on RECs.

Increased mucociliary differentiation of human respiratory epithelial cells on hyaluronan-derivative membranes.

The selection of a scaffold to facilitate mucociliary differentiation of respiratory epithelial cells (RECs) is crucial in the development of tissue engineering of respiratory epithelium. However, how the differentiation of RECs is influenced by the biomaterials has never been thoroughly explored. Previously, hyaluronan derivatives were considered as unsuitable biomaterials for culture of respiratory epithelium. In contrast, this study demonstrates that the membranous scaffolds made from benzyl esters of hyaluronic acids are capable of providing a more preferential environment for human RECs than conventionally used collagen-based scaffolds. The proliferation and mucociliary differentiation of RECs were examined by MTT assays, scanning electron microscopy, immunofluorescence, immunoblotting and gene expression. The percentage of ciliated cells in cultured RECs increased from 12.4% on collagen to 20.4% on hyaluronan-derivative membranes with a pseudostratified polarized layer that closely resembled the composition of the native epithelium. The expression levels of MUC5AC and MUC5B mRNA were higher on hyaluronan-based scaffolds than those on collagen. The presence of a hyaluronan-binding domain, CD44 and the receptor for hyaluronan-mediated motility of RECs were also demonstrated. Accordingly, the mucociliary differentiation-promoting effect of hyaluronan-derivative membranes indicates that it may be applied to the tissue engineering of respiratory epithelium.

Human salivary gland acinar cells spontaneously form three‐dimensional structures and change the protein expression patterns

Applying tissue engineering principles to design an auto‐secretory device is a potential solution for patients suffering loss of salivary gland function. However, the largest challenge in implementing this solution is the primary culture of human salivary gland cells, because the cells are highly differentiated and difficult to expand in vitro. This situation leads to the lack of reports on the in vitro cell biology and physiology of human salivary gland cells. This study used a low‐calcium culture system to selectively cultivate human parotid gland acinar (PGAC) cells from tissues with high purity in cell composition. This condition enables PGAC cells to continuously proliferate and retain the phenotypes of epithelial acinar cells to express secreting products (α‐amylase) and function‐related proteins (aquaporin‐3, aquaporin‐5, and ZO‐1). Notably, when the cells reached confluence, three‐dimensional (3D) cell aggregates were observed in crowded regions. These self‐formed cell spheres were termed post‐confluence structures (PCSs). Unexpectedly, despite being cultured in the same media, cells in PCSs exhibited higher expression levels and different expression patterns of function‐related proteins compared to the two‐dimensional (2D) cells. Translocation of aquoporin‐3 from cytosolic to alongside the cell boundaries, and of ZO‐1 molecules to the boundary of the PCSs were also observed. These observations suggest that when PGAC cells cultured on the 2D substrate would form PCSs without the help of 3D scaffolds and retain certain differentiation and polarity. This phenomenon implies that it is possible to introduce 2D substrates instead of 3D scaffolds into artificial salivary gland tissue engineering. J. Cell. Physiol. 226: 3076–3085, 2011.

Formation of post-confluence structure in human parotid gland acinar cells on PLGA through regulation of E-cadherin.

As a potential solution for patients to retrieve their lost salivary gland functions, tissue engineering of an auto-secretory device is profoundly needed. Under serum-free environment, primary human parotid gland acinar (PGAC) cells can be obtained. After reaching confluence, PGAC cells spontaneously form three-dimension (3D) cell aggregations, termed post-confluence structure (PCS), and change their behaviors. Poly (lactic-co-glycolic acid) (PLGA) has been widely used in the field of biomedical applications because of its biodegradable properties for desired functions. Nonetheless, the role of PLGA in facilitating PGAC cells to form PCS has seldom been explored to recover epithelial characteristics. In this study, PGAC cells were found to have a greater tendency to form PCS on PLGA than on tissue culture polystyrene (TCPS). By tracing cell migration paths and modulating E-cadherin activity with specific inhibitor or antibody, we demonstrated that the static force of homophilic interaction on surfaces of individual cells, but not the dynamics of cell migration, played a more important role in PCS formation. Thus, PLGA was successfully confirmed to support PGAC cells to form more PCS through the effects on enhancing E-cadherin expression, which is associated with FAK/ILK/Snail expression in PGAC cells. This result indicates that selective appropriate biomaterials may be potentially useful in generating 3D PCS on two-dimension (2D) substrate without fabricating a complex 3D scaffold.

Clinical and biomechanical analyses to select a suture material for uvulopalatopharyngeal surgery

OBJECTIVE The selection of the degradation pattern of suture materials is critical in uvulopalatopharyngeal surgery because of the complex oral environment such as the constant presence of saliva; the microbial accumulation; and the functions related to speech, mastication, and swallowing. The aim of the study was to identify appropriate suture materials for uvulopalatopharyngeal surgery by analyzing the clinical and biomechanical results obtained using four different absorbable suture materials. STUDY DESIGN A randomized open-label trial. SETTING Tertiary referral center. SUBJECTS AND METHODS Eighty patients with obstructive sleep apnea syndrome who had undergone microdebrider-assisted extended uvulopalatoplasty were randomized into the four suture groups: sutures of polyglycolide (Dexon) and copolymers of polyglycolide and other degradable polymers (Polysorb, Monocryl, Maxon). Handling characteristics, surgical outcomes, Youngs modulus reduction, morphologic changes by scanning electron microscope, and saliva absorption test were analyzed. RESULTS Monocryl (poly(glycolide-co-∊-caprolactone)) had the optimal handling characteristics and exhibited two-stage degradation: sufficient tensile strength was maintained initially to allow wound healing, and then the suture degraded rapidly to avoid irritation. In contrast, the hydrolytic degradation of Dexon, Polysorb, and Maxon seemed to involve only an approximately single-stage process in the oral environment. Moreover, a large amount of debris was observed among filaments in Dexon and Polysorb, whereas only some scattered debris accumulated on Monocryl and Maxon under scanning electron microscope. CONCLUSION Poly(glycolide-co-∊-caprolactone) has good handling characteristics and degrades in two stages. It is a suitable suture material for uvulopalatopharyngeal surgery.

Preliminary report of vocal fold augmentation with cross-linked porcine collagen.

Objective. To evaluate the short-term outcomes of using cross-linked porcine collagen for vocal fold augmentation. Study Design. Case series with planned data collection. Setting. Tertiary referral hospital. Subjects and Methods. Twenty consecutive patients of unilateral vocal fold paralysis (UVFP) were recruited, including 10 males and 10 females. All the subjects received videolaryngostroboscopic (VLS) evaluation for the glottic closure pattern, perceptual grading using the GRB scale (grade, roughness, breathiness), acoustic analysis, maximal phonation time (MPT), and Voice Handicap Index (VHI) questionnaire. Purified telopeptide-free, glutaraldehyde cross-linked porcine collagen was injected under local anesthesia with fiberscopic guidance. Results. Treatment outcomes were evaluated 3 months after the injection. Fifteen (75%) of 20 patients reported complete resolution of symptoms, whereas the other 5 (25%) patients had substantial improvements. The median score of perceptual analysis of voice quality showed significant improvements from G2/R2/B2 to G1/R1/B0 (P < .001). Glottic closure pattern under stroboscopy had improved from “predominantly open” before the procedure to a “half-open and half-close” pattern after the procedure (P < .001). Acoustic analysis demonstrated significant improvements of jitter and normalized noise energy (P < .05). Maximal phonation time revealed a significant improvement from 7.5 ± 3.2 seconds to 13.1 ± 3.9 seconds (P < .01). The VHI had also decreased from 53 ± 25 points to 27 ± 20 points (P < .01). No patients had serious adverse events during the follow-up period. Conclusion. This case series demonstrated that porcine collagen may be a suitable material for temporary vocal fold augmentation.

Increased mucociliary differentiation and aquaporins formation of respiratory epithelial cells on retinoic acid-loaded hyaluronan-derivative membranes

While playing a major role in maintaining the mucociliary phenotype of respiratory epithelial cells (RECs), retinoids are critical determinants of their normal function. However, despite being a powerful biological agent, retinoic acid (RA) is generally not used in regenerative medicine due to its scarce bioavailability via conventional administration. Therefore, the ability to incorporate RA into biomaterials allows for a combination of the biological effects of RA and biomaterials in influencing cellular behavior. This study attempts to develop RA-loaded hyaluronan-derivative membrane (RA-HAm) and investigates how this membrane affects the mucociliary differentiation and aquaporins (AQP) formation of RECs. In a simulated in vitro culture condition, the RA release from membranes is maintained for 7days. On the seventh day, the cumulative release rate of RA from supportive biomaterials is ~87% under detect limitation. RECs cultured on RA-HAm reveal numerous mature ciliated cells and microvilli compared to aggregated cilia-like structures on hyaluronan-derivative membrane (HAm). Moreover, the expression levels of MUC5AC and AQP on RA-HAm are higher than those on HAm. The proposed model elucidates the release of hydrophobic RA from hyaluronan-derivative biomaterials. We believe that RA-loaded hyaluronan biomaterials are highly promising biomaterials for use in sinonasal surgery and tissue engineering of the respiratory system.

Selective culture of different types of human parotid gland cells.

Advances in salivary gland tissue engineering can benefit patients diagnosed with xerostomia. Complexity of the gland explains the urgent demand for a reliable protocol to isolate and expand various gland cells that can be used for further study.

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 βIII 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.