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 cells. The cell proliferation profile was similar for both TG-IGF1 and native recombinant IGF1. Although we were able to obtain relatively pure TG-IGF1 protein, the purification protocol may still require some development due to loss of protein via aggregation during protein production. Using a three-dimension in vitro model, neonatal human bladder smooth muscle cells were seeded in the fibrin gel only, in the fibrin gel either with TG-IGF1 or native recombinant IGF1 as a control. The morphology of the seeded cells was analysed using ultra-structural transmission electron microscopy after three days. Secretory vesicles had not been found in the cells without IGF1. The cells with the TG-IGF1 displayed larger and more numerous secretory vesicles than those with native recombinant IGF1, presumably due to the rapid diffusional loss of the native recombinant IGF1 from the gel. The bladder smooth muscle cell in the fibrin gel responses to either TG-IGF1 or native recombinant IGF1 at the genomic level were analyzed by qRT-PCR for extracellular matrix and adhesion molecules after 24 hours incubation. The cells in the fibrin gel only were used as control. We did not observe a difference in gene expression between the two groups. The bioactivity of TG-IGF1 was also investigated in vivo utilizing a rat bladder model. A wound was induced via resection on the rat bladder. Either the TG-IGF1 or native recombinant IGF1 containing fibrin gel was then applied to the wound site, with a second control group receiving only the fibrin gel alone. Three classic histological stainings were done using Hematoxylin & Erythrosine (HE), Masson's Trichrome (TM) and Prussian Blue (PB). It was found that TG-IGF1 greatly enhanced rat bladder muscle layer regeneration compared to native recombinant IGF1 and the control group of fibrin gel according to the histology staining and quantitative analysis of the ratio of detrusor muscle regenerated / normal detrusor muscle (0.27±0.10 for TG-IGF1 and 0.23±0.10 for native recombinant IGF1). The second part of the work was devoted to making a new generation of fibrin matrices. Fibrinogen (Fgn), a soluble plasma protein found in all vertebrates, is a covalent dimer composed of pairs of three polypeptide chains called Aα-, Bβ- and γ-chains. Fibrinogen Aα chain can be truncated to Bβ-and γ-chains while maintaining the capacity to be assembled into a secreted, detectable fibrinogen. It is feasible to produce recombinant chimeric fibrinogen in cell culture, which can then be used to incorporate growth factors into fibrin matrices as a fusion protein with fibrinogen at the genetic level. In this way, fibrin based natural material hydrogels can be formed which can bind and release signalling biomacromolecules for directed cellular growth and tissue regeneration. In order to produce this new fibrinogen recombinant protein, a novel stable cell line of Chinese Hamster Ovary cells, CHOfgn-hIGF1, needed to be developed. This was accomplished via transfection of an FGA-hIGF1 plasmid DNA into CHOβγ cells. After protein production, the recombinant chimeric fibrinogen variant hIGF1 fusion protein was purified by affinity chromatography. The successful production of these cell lines and the resultant variant IGF1 was confirmed via ELISA with fibrinogen and hIGF1 antibodies, and also by fluorescence activated cell sorting (FACS). Due to protein polymerisation in the column during purification, the protein purification step still requires additional development. Although the targeted fusion protein was successfully produced, overall protein yield was so low as to prevent further exploration. By harnessing the powerful technique of protein engineering and using the fibrinogen/fibrin hydrogels developed in our lab, we were able to demonstrate a new type of multifunctional gel capable of regenerating smooth muscle type tissue with the aim to repair bladder function. Here we were able to demonstrate the production of the variant IGF1 factor XIIIa substrate fusion protein, its biological activity in vitro, and its application in an in vivo rat model.
EPFL2010
