Tissue engineering of a tracheal substitute

Lectin histochemistry and scanning electron microscopy (SEM) was
used to assess the growth and characterise the differentiation of
human respiratory epithelial cells (REC) cultured on two
biomaterial scaffolds. The first scaffold, based on a hyaluronic
acid derivative, was observed to be non-adhesive for REC. This lack
of adhesion was found to be unrelated to the presence of the
hyaluronic acid binding domain on the surface of isolated REC. The
other scaffold, consisting of equine collagen, was observed to
encourage REC spreading and adhesion. Positive Ulex Europaeus
agglutinin (UEA) lectin staining of this preparation indicated the
presence of ciliated REC on the scaffold surface. However, the
marked decrease in peanut agglutinin (PNA) positive staining,
relative to that of control cultures and native tissue, indicates a
dedifferentiation of the secretory cells in monolayer. SEM analysis
of REC cultured on the collagen scaffold confirmed the presence of
ciliated cells thereby validating the UEA positive staining. The
presence of both established and developing cilia was also
verified. This indicates that collagen biomaterials are appropriate
for the tissue engineering of REC. Furthermore, that UEA and PNA
staining is a useful tool in the characterisation of cells cultured
on biomaterials, therefore helpful in identifying biomaterials that
are suitable for specific tissue engineering purposes. The culture
of REC at an air liquid interface (ALI) was investigated. Both
conventional ALI inserts and the Biofleece scaffold were used. The
cells grown the on conventional inserts became multilayered and
showed some degree of ciliation after the period of ten days. The
cells grown on the Biofleece scaffold became necrotic and died due
to nutrient deprivation. The use of ALI culture techniques on
scaffold materials needs to be adjusted to allow for sufficient
nutrient supply to the cells. The Biofleece scaffold was found to
be suitable for the tissue engineering of cartilage in vitro.
Constructs with a cartilage-like morphology were generated with the
scaffold after two weeks in culture. The tissue-engineered
cartilage was found to contain a higher number of cells and less
extracellular matrix (ECM) than the native tissue controls. Suction
seeding techniques were used to improve the distribution of cells
within the scaffold and thereby increase the overall efficiency of
cartilage tissue engineering within the scaffold. Alcian blue (AB)
and Papanicolau (PN) stains of the tissue engineered cartilage
described two distinct regions within the constructs, namely the
developed cartilage-like region and the developing region. The
latter is thought to be areas in which the cartilage cells are yet
to fully remodel the scaffold material and deposit their own
“native” ECM. However, the Biofleece scaffold material was observed
to loose 40-50% of its initial volume during the tissue engineering
process over a period of two weeks. Thus the degradation of the
Biofleece scaffold exceeds the rate of maturation of the cartilage
tissue within the scaffold. This rapid biodegradation is most
likely a result of matrixmetalloproteinase (MMP), in particular
collagenase, production by the maturing chondrocytes. This
reduction in size means that the Biofleece scaffold is not an
appropriate material for the tissue engineering of a trachea. The
optimal biomaterial for the tissue engineering of a trachea would
degrade at a rate equal too, or slower than, the time taken for the
cells within the scaffold to mature into functional tissue. The
co-culture of REC and chondrocytes was achieved through the use of
matrigel as a basement membrane replacement (note that direct
growth of REC on cartilage tissue has been observed to be
difficult). The co-cultured constructs were not stable because the
Biofleece scaffold degrades at a high rate in the presence of both
cell types. The constructs were observed to shrink to approximately
35-30% of the original dimensions in a period of 3-7 days. The
reason for this accelerated degradation is not known but is most
likely the result of severe MMP production by the two cell types
when in combination. It was concluded that the characterisation
procedures used in this study (histochemical staining, fluorescent
staining and scanning electron microscopy) for both REC and
chondrocyte tissue engineered constructs are appropriate for this
and further studies. The chondrocyte seeding methodologies in
particular are a useful tool for tissue engineering. This study
succeeds in many ways to investigate the tissue engineering of a
tracheal substitute by detailing how REC and chondrocytes can be
cultured on biomaterials and assessed for tissue development.
However, the study does not deliver such a viable substitute as an
end product. The primary reason for this outcome is the rapid
degradation of the Biofleece scaffold material