Publication
Currently, both congenital abnormalities and developmental problems of the bladder in children, and other dysfunctions in adults, require reconstructive surgery. Such correction involves transplant action of native tissues (such as gastrointestinal segments, or mucosa), homologous tissues from a donor, heterologous tissues or substances, or artificial materials to act as a replacement for normal bladder tissue. However, such surgery does not entirely restore the function, as the replacement tissue is either rejected due to immune system, fibrosis, contraction or causes metabolic complications due to a mismatch in different functional parameters, such as gastrointestinal segment for absorbance versus bladder for excretion. Tissue engineering is emerging as a significant alternative potential treatment for bladder dysfunction. To achieve this goal, we have explored fibrin gels as a natural material based scaffold to seed infiltrating smooth muscle cells to mimic the ontogeny of the bladder tissue. Our interest in developing fibrin-based biomaterial technology is based on fibrin's central role in tissue binding and in the initiation of tissue repair and defence. It is well known that fibrinogen/fibrin binds to platelets as well as to different cells, growth factors, and extracellular matrix proteins, which is critical for the wound repair process. Insulin-like growth factor I (IGF1) is well known as a key regulator in carbohydrate metabolism and growth. It can promote smooth muscle cell growth. In this thesis it was also chosen due to its simple active form, namely a single chain of low molecular weight, 10kDa. A novel engineered insulin-like growth factor I (IGF1) – factor XIIIa substrate fusion protein was chosen as the bioactive macromolecule to be released from the scaffold in order to control cellular growth and differentiation. Therefore, we can achieve close to the natural biomechanical environment in the regenerated tissue. In this work, two generations of fibrin matrices for tissue engineering were developed. The first part of the work was devoted to preventing rapid diffusion of the growth factor from the matrix and to controlling its release. IGF1 was modified by inserting a factor XIIIa substrate sequence (denoted TG) based on the α2-plasmin inhibitor to cross-link into the fibrin gel during coagulation. This is so that the variant IGF1 will bind to the fibrin gel itself and will be released upon matrix degradation. In order to produce this recombinant protein TG-IGF1, IGF1 GST tag plasmid DNA was transformed into BL21 competent cells. After protein production, the recombinant TG-IGF1 protein was purified by GST affinity chromatography. The purified TG-IGF1 was confirmed via SDS-PAGE and western blot with an anti-hIGF1 antibody. The sequence was confirmed using MALDI-TOF mass spectrometry. The biological activity of TG-IGF1 was validated in vitro by receptor tyrosine phosphorylation and metabolic assay (MTT assay) with fibroblast 3T3 cel