The standard approach of tissue engineering is to develop a cell-seeded scaffold
which has morphology and mechanical properties similar to those of the target tissue.
The scaffold material should possess surface properties so that the seeded cells can
maintain proper activity, function and morphology. In terms of engineering of
pulmonary epithelial tissue, a major challenge is to obtain sufficient cells because of
the difficulty in isolating and culturing primary pulmonary epithelial cells in vitro,
especially type II pneumocytes. To overcome this problem, a method to differentiate
pulmonary progenitors, which possessed most of the features of type II pulmonary
epithelial cells, from murine embryonic stem cells (mESCs) was investigated in this
study. The first aim of this project was to increase the differentiation efficiency of type
II pneumocyte progenitors from mESCs by enhancing the efficiency of an established
differentiation protocol using extracellular matrix (ECM), growth factors and
bioactive peptides. In the first study, mESCs were differentiated on tissue culture
plastic (TCP) and poly(D,L-lactide) (PDLLA) films coated with ECM proteins, collagen, laminin, fibronectin and Matrigel. The results demonstrated that
all protein coatings can enhance the wettability of the TCP and PDLLA films and,
moreover, laminin and Matrigel can enhance the differentiation of mESCs into
pulmonary progenitors. In the second study, growth factors that are commonly
thought to affect the development of embryonic lung, including fibroblast growth
factors (FGFs) 1, 2, 7 and 10, were added to the differentiation culture at various
concentrations and the subsequent expression of surfactant protein C, a marker of type
II pneumocytes and their progenitors, was measured. It was found that FGF1 alone
and FGF10 in combination with Matrigel coating enhanced the differentiation of
mESCs into pulmonary progenitors. In another study, mESCs were differentiated on
PDLLA films grafted with the bioactive peptides RGD and YIGSR. Preliminary result
showed that YIGSR enhanced the differentiation of mESCs into pulmonary
progenitors.
The second aim of this project was to develop 2D environments and 3D scaffolds
made of PDLLA suitable for the culture of human pulmonary epithelial cells (A549
line). PDLLA has advantages of biocompatibility and biodegradability, but a major drawback is its hydrophobic nature. To make the surface of PDLLA films hydrophilic,
it was modified using a variety of methods, i.e. by grafting the bioactive peptides
RGD and YIGSR, by introducing amines using aminolysis and by creating
amine-terminated and carboxylic acid-terminated tree-like branched architectures on
to the surface. The surface properties of modified PDLLA films were evaluated using
various techniques. The culture of A549 cells on PDLLA films demonstrated that
surface modifications can affect the attachment, focal adhesion point formation,
activity and population size, depending on the type and the concentration of the
bioactive peptides or functional groups presented on the surface of PDLLA films.
The challenge of culturing pulmonary epithelial cells in 3D is to generate scaffolds with proper porous structures which allow sufficient medium diffusion in and waste
disposal out of the scaffolds. The influence of the preparation conditions, i.e.
coarsening time and coarsening period of a liquid-liquid phase separation system and
freezing temperature of a solid-liquid phase separation system, on the porous
morphology and the subsequent pulmonary epithelial cell culture were examined.
Scaffolds that possessed alveolus-like round pores and ladder-like pores were
prepared using liquid-liquid phase separation and solid-liquid phase separation,
respectively. Culture of A549 cells on the PDLLA scaffolds demonstrated that cell
penetration into and activity on the scaffolds are influenced by the pore size and the
pore throat size of the scaffolds.
In conclusion, the results of this project demonstrated that the differentiation of
mESCs into pulmonary epithelial progenitors can be enhanced by external signals i.e.
from ECM proteins, FGFs and bioactive peptides. The responses of pulmonary
epithelial cells to the PDLLA scaffolds can be enhanced by surface modifications
using bioactive peptides and functional groups and scaffolds that can serve as a
culture environment for pulmonary epithelial cells were prepared accordingly. Taken
together, the results of these studies provide a basis for future engineering of
pulmonary epithelial tissue, an area of tissue engineering that lags behind that of other
major organs.