| Summary: | 博士 === 國立臺灣大學 === 醫學工程學研究所 === 107 === Cell proliferation or extracellular matrix secretion is necessary for human tissues. Due to slow proliferating properties of cells in vivo, or insufficient extracellular secreting, some tissues cannot automatically repair after damage. Thus, we want to develop some biomaterials to improve the repairability of those tissues.
In this study, we focused on neurons and corneal endothelial cells and tried to improve existing biomaterials for tissue engineering. For neurons, (N-(4-aminobutyl)-acrylamide) (P4Am) is a positively charged material similar to well-known ploy-D-lysine(PDL), which is regularly used for neuron culture. The main difference is the peptide structure, which is in the backbone of PDL but locating at the side chain of P4Am. We assumed that neurons are sensitive enough to distinguish such structure difference, so these two cationic polymers were compared at serial coating concentrations for culturing cerebellar granule neurons from 7-day-old Wistar rats in this study. Cellular viability and morphology assay in the peptide structure between P4Am and PDL could be distinguished by neurons at low coating concentrations (< 0.16 µg/ml). P4Am at low coating concentration could keep aggregates with three or four thick processes to support more complete neural network with higher cellular viability than PDL. This suggests that the interaction between neurons and the specific peptide structure of P4Am at low coating concentration was able to improve survival and differentiation of cultured cerebellar granule neurons.
Although mature neurons cannot proliferate, new material increased cell viability and network connection. For severe neural damage, guiding neurite outgrowth and maintaining networks are essential. Therefore P4Am is a potential candidate for neural tissue engineering.
For cornea, we aimed to investigate the underlying mechanisms of the differentiation corneal endothelial cells (CECs) and to identify the compositions of extracellular matrix (ECM) using a chitosan/ polycaprolactone (PCL) blended membrane to explore the potential use of chitosan/PCL blends in tissue engineering of CECs. Bovine CECs were cultured on the blends and compared with TCPS and pure chitosan membrane. Cell behaviors in terms of cell attachment, proliferation, differentiation phenotype and expression of differentiation proteins were examined. Furthermore, the production of ECM proteins was also analyzed. Through experiments, we found that the topography (roughness) of PCL25 membrane examined by AFM was greater than that of pure chitosan membrane. FTIR results confirmed the C=O groups of PCL. The CECs displayed hexagonal morphology and a similar proliferation rate on both PCL25 membrane and TCPS. The production of ECM protein productions of CECs on PCL was not inferior to TCPS. Moreover, Western blot results verified the higher amount of collagen IV, and lower TGF-β2 expression on PCL25 membrane than that on TCPS substrate.
Although the nervous system and corneal endothelial cells hardly completely recover in the human body, our studies found not only a potential material but also new indicators to qualify our materials for tissue engineering.
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