Biofunctional Scaffold Design for Soft Tissue Regeneration

Fibrin has long been used clinically for hemostasis and sealing and has been extensively characterized as a biomaterial. Yet, in terms of harnessing its potential for use in tissue regeneration, there remains a wide gap between in vitro experimentation and clinical application. Important factors contributing to this disjunction are rapid resorption in vivo, handling capability, and therapeutic robustness. This thesis addresses some of the shortcomings associated with the use of fibrin in tissue regeneration by offering clinically feasible solutions to improve fibrin biomaterials as regenerative therapies. We sought to improve the pharmacokinetic profile of fibrin in vivo by modifying aprotinin, a small protease inhibitor commonly contained within clinical fibrin sealant formulations to slow gel degradation. In exploiting a platform technology from the Hubbell laboratory, we created an aprotinin variant which covalently binds to fibrin during normal thrombin- and factor XIIIa-mediated crosslinking. This mutant protein successfully extended the longevity of fibrin by supplying local reservoirs of protease inhibitor at fibrin's naturally evolved inhibitor-binding sites. More specifically, we demonstrated that our aprotinin variant is able to prolong the life of fibrin by at least 3-fold both in in vitro and in vivo settings. However, clinical objection to aprotinin has arisen in recent years relating to concern over sourcing and immunogenicity due to its animal origin. We thus sought to apply the same protein modification scheme to an analogous human sequence derived from the Kunitz Protease Inhibitor (KPI) domain of the amyloid beta (A4) protein. In challenging the resultant mutant with physiological plasmin levels in vitro, we have established that the KPI variant extends the life of fibrin matrices by approximately 2-fold longer than wild type aprotinin. Currently, efforts are underway to improve the quality of KPI variant production for further in vitro and in vivo characterization. Additionally, we focused efforts on improving the handling capability of fibrin matrices through integration with polymer mesh or collagen sponge. By reinforcing fibrin matrices with these commercially available materials, we created hybrid matrices that are easy to handle and readily suturable. Combination of these materials facilitates a more comprehensive range of in vivo applications for which fibrin is suitable in soft tissue engineering. Finally, we sought to increase the therapeutic robustness of fibrin through covalent immobilization of human insulin-like growth factor-1 (IGF-1) within the matrix. Again, we employed the same protein modification scheme to create an IGF-1 variant which covalently crosslinks to fibrin during normal polymerization. Through in vitro and in vivo models, we established that sustained presence of the IGF-1 variant elicits a more prolonged and robust response from bladder smooth muscle cells than the wild type form of the protein. In conclusion, this work highlights clinically viable solutions to address several of the current shortcomings of fibrin for use in in vivo applications. By incorporating innovatively designed components, fibrin can be translated into a biofunctional scaffold and effective therapeutic for soft tissue regeneration.

Hydrogels for Urinary Tract Tissue Engineering

Lionel Ambroise Micol

Several urinary tract disorders, such as hypospadias or bladder cancer, require reconstructive surgery to preserve the patient's quality of life and at times even save their life. The current available surgical treatments, involving grafts from other tissue origin, can, however, lead to long-term complications in up to 50% of these patients. New alternatives are therefore considered among which tissue-engineered grafts are thought to be the most promising approach. Tissue-engineered grafts have so far been shown to require the presence of cells, which involves different steps prior to implantation including cell isolation and expansion, seeding on a matrix, and further culture. These steps are time-consuming, costly, and raise safety concerns regarding the in vitro cultured cells. In this thesis, we sought to further improve some tissue-engineered grafts with approaches that would either reduce or replace the cell culture steps. One of these approaches relied on the plastic compression of collagen gels. Ready cell-seeded plastic compressed collagen gels can be rapidly formed within minutes and have much better mechanical properties than regular collagen gels that are known to be fragile. In order to have a stronger gel we adapted the plastic compression concept and developed high-density collagen gels tubes (hdCGTs) for urinary tract tissue-engineering. We demonstrated that they were suitable for smooth muscle cell (SMC) proliferation and that their mechanical response to dilation and extension were compatible with ureter and urethra physiologic values. Hence, these ready-seeded hdCGTS represent promising tissue-engineered constructs that can shorten the production time of cell-seeded grafts for urinary tract regeneration. We further tested our hdCGTs in a rabbit model to investigate their regenerative capacity in vivo. Autologous rabbit SMCs were isolated from a bladder biopsy to form cellular constructs, while acellular constructs were used as control. Both types of constructs were sealed with fibrin glue at the site of a newly created urethral defect. Rabbits were evaluated by retrograde urethrography after 1 or 3 months. Despite a high rate of fistula and a relative narrowing of graft calibers, all rabbits survived and showed micturition within 1-3 days following surgery. SMC-seeded grafts showed, however, a better outcome at 3 months in accordance to literature. Since plastic compressed collagen gels are still too weak to be sutured, we decided to strengthen them by combining them with a polymer mesh. We investigated flat collagen-poly(lactic acid-co-ε-caprolactone) (PLAC) hybrid scaffolds seeded with human bladder cells for their behaviour in the subcutaneous space of nude mice. Host cell infiltration, scaffold degradation, and the presence of the seeded bladder cells were analyzed at different time points on a total period of 6 months. The hybrids showed a lower inflammatory reaction in vivo than PLAC meshes alone, and first signs of polymer degradation were visible at six months. The collagen-PLAC hybrids have potential for urinary tract tissue regeneration as they show efficient cell seeding and proliferation as well as proper mechanical properties for surgical handling. Another approach towards improving tissue-engineered grafts was to use off-the-shelf smart biomatrices to replace the cell culture steps. This involved the development of cell-controlled release of insulin-like growth factor-1 (IGF1) in an acellular collagen matrix. IGF1 induces SMC migration, proliferation, and differentiation, and can hence promote the invasion and population of the acellular matrix by cells in the surrounding tissue once implanted in vivo. In this approach, we designed and produced a recombinant collagen-binding IGF1. The IGF1 domain would then be released after cleavage from an affinity binding domain to collagen under the action of cell-secreted matrix metallo-proteases (MMP). Although our recombinant IGF1 showed the expected bioactivity on SMCs and sensitivity to MMP-2, its affinity for type I collagen (Col-I) could not be demonstrated. We further investigated covalent binding alternatives involving transglutaminase (TG) crosslinking or IGF binding protein (IGFBP)-mediated binding to collagen. TG crosslinking did not allow for sufficient amount of a TG-IGF, a recombinant form of IGF1 presenting a substrate sequence for plasma TG, to be bound to Col-I. The entrapment of IGFBP5 within plastic compressed collagen gels however suggests that collagen compression and collagen-binding means could be combined in order to allow for a more controlled-release profile of IGF1 from collagen gels. Lastly, we sought to develop a new bioengineered solution for bladder sphincter disorders, such as urinary incontinence or vesico-ureteral reflux. These disorders are treated with open surgeries susceptible of complications and extended hospital stay. Endoscopic treatment by injection of bulking agents is thus usually preferred, although they often necessitate additional interventions for a sustained effect. With the idea to suppress this need for additional intervention and thus to limit discomfort for patients as well as costs, we sought for a permanent bulking agent. We therefore developed an injectable poly(acrylonitrile) hydrogel paste injected into the submucosal space of pig bladders. The implants were harvested at days 7, 14, 21, 28, 84, and 168 and analyzed morphologically and by histology. The persistence of the implants was demonstrated, indicating that this injectable bulking agent could potentially be used as biomaterial for the treatment of urinary incontinence and vesico-ureteral reflux.

EPFL2011

IGF-1-containing multi-layered collagen-fibrin hybrid scaffolds for bladder tissue engineering

Priscilla Suhasna Maithili Briquez, Peter Frey, Jeffrey Alan Hubbell, Hans Mattias Larsson, Kalitha Pinnagoda, Elif Vardar

Clinical success of bladder reconstructive procedures could be promoted by the availability of functional biomaterials. In this study, we have developed a multi-layered scaffold consisting of a bioactive fibrin layer laminated between two collagen sheets all having undergone plastic compression. With this construct we performed bladder augmentation in a nude rat model after partial bladder excision and evaluated the morphological and functional behavior of the implant. The fibrin was functionalized with a recombinant human insulin-like growth factor-1 (IGF-1) variant that covalently binds fibrin during polymerization and has a matrix metalloproteinase-cleavage insert to enable cell-mediated release. The purified IGF-1 variant showed similar bioactivity in vitro compared to commercially available wild type (wt) IGF-1, inducing receptor phosphorylation and induction of human smooth muscle cell proliferation. In vivo, the multi-layered bioactive collagen-fibrin scaffolds loaded with the IGF-1 variant triggered dose dependent functional host smooth muscle cell invasion and bundle formation with re-urothelialization 4 weeks after surgery in a rat model.

Elsevier Sci Ltd2016

Rat bladder muscle regeneration induced by local delivery of an engineered insulin-like growth factor 1 in fibrin gels

Lirong Yang

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.