Tissue engineering of a tracheal substitute (2004)

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