The applications of biomaterials in tissue engineering of respiratory epithelium
Date Issued
2010
Date
2010
Author(s)
Huang, Tsung-Wei
Abstract
Comprising mucociliary epithelium, respiratory epithelium serves as an important defense mechanism against inhaled toxins, pathogens, and particles. Discontinuity of the epithelium, e.g., CSF rhinorrhea, may cause headache and central nervous system infection. Septal perforation may cause inflammation, nasal crusting, bleeding, and whistling while breathing. To date, various surgical repairs for the septal perforation are proposed, such as advancement mucosal flaps, lateral nasal wall flaps, and autografts using temporal fascia. However, several drawbacks are encountered, i.e., donor site morbidity, tissue shortage, and retention of the original characteristics of the donor tissue. Therefore, several biocompatible biomaterials are developed to serve as grafts. Most graft materials are composed mainly of collagen, because collagen can promote epithelial growth and mucociliary differentiation. However, several disadvantages of collagen, e.g., fast biodegrading rate, low mechanical strength, and extremely high cost, restrict its clinical usage. Chitosan, like glycosaminoglycan in extracellular matrix, is characterized by its low cost, biodegradable, nonantigenic, and tailorable properties. Nevertheless, whether chitosan is exploited as a scaffold for growth and differentiation of the nasal epithelium remains unclear. The aim of this study is first to evaluate whether chitosan-based membranes can be used for culturing respiratory epithelial cells (RECs) and investigate their effect on mucociliary differentiation.
RECs are cultured on three various substrates, e.g., chitosan membranes, collagen, and chitosan-collagen membranes. Morphology of RECs is examined via light and electron microscopy, the area of ciliated cells is measured by confocal microscopy. Expression of mucin genes is investigated with reverse-transcription polymerase chain reaction. RECs are found to be successfully adhesive with collagen and chitosan-collagen membranes at day 3 after seeding, but not with chitosan membranes. The cilia area on collagen is non-significantly different from that on chitosan-collagen membranes. The expression levels of mucin genes, namely, MUC5AC, MUC5B, and MUC2, in RECs on both collagen and chitosan-collagen membranes do not differ significantly. This study demonstrates that a small amount collagen mixed with chitosan substrate may improve the biocompatibility and promote the mucociliary differentiation in RECs. It appears that chitosan-collagen membrane is a promising scaffold for culture of the nasal epithelium and can be applied for repairing nasal mucosa defect in the future.
In addition, how to repair extensive respiratory tract defect after resecting trachea or bronchus due to tumor or tuberculosis remains a challenge. The new therapeutic strategy has been suggested using tissue engineered airway replacement. A key issue for this strategy is the selection of biomaterials which can facilitate growth and differentiation of RECs. Hyaluronan, one of the chief components of the extracellular matrix, contributes significantly to cell proliferation, migration, and differentiation. In the respiratory system, hyaluronan has been proposed to serve a pivotal role in mucosal host defense by stimulating ciliary beating of RECs. Therefore, we hypothesize that hyaluronan may serve as the ideal scaffold for tissue engineering of respiratory epithelium.
However, unmodified hyaluronan is found only in gel form and possesses a very short degradation time. For it to be used in tissue engineering, it would need to be chemically modified to improve its structural properties and increase its resistance to degradation. 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 (HYAFFR) 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. Although MTT reveals that RECs on collagen have higher proliferation rates than that on HYAFF, HYAFF promotes more ciliary differentiation of RECs than collagen. The percentage of ciliated cells in cultured RECs increases from 12.4% on collagen to 20.4% on HYAFF with a pseudostratified polarized layer that closely resembles the composition of the native epithelium. The expression levels of MUC5AC and MUC5B mRNA are higher on HYAFF than those on collagen. The presence of a hyaluronan-binding domain, CD44 and the receptor for hyaluronan-mediated motility (RHAMM) of RECs are 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.
However, the regulatory mechanism of ciliary differentiation-promoting effect of hyaluronan-based biomaterials remains unknown. Hyaluronan influences proliferation and differentiation of various cells through two main receptors, CD44 and RHAMM. RHAMM remains the most poorly understood hyaluronan receptor as it can act as a cell surface receptor but it can also be localized in the cytoplasm, cytoskeleton, or in the cell nucleus, displaying both hyaluronan dependent and independent functions. Additionally, RHAMM also functions as a microtubule-associated protein interacting with microtubules, which are the components of cilia. Therefore, it is anticipated that RHAMM may play a role in regulating the ciliary differentiation of RECs on hyaluronan-based biomaterials. Additionally, based on the literature, retinoic acid (RA) is commonly used as a constant medium supplementation to promote ciliary differentiation of RECs in vitro. The aim of the study is to investigate the ciliary differentiation of RECs on HYAFF with/without RA compared with that on collagen with/without RA and further to elucidate the role of 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. RECs on collagen with RA also express higher level of RHAMM mRNA than those 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.
Subjects
Chitosan
Collagen
Hyaluronan
Respiratory epithelial cells
Mucociliary differentiation
Retinoic acid
Receptor for hyaluronan-mediated motility (RHAMM)
SDGs
Type
thesis
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