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.

Hubbell, Jeffrey A.
Frey, Peter
Lausanne, EPFL
Other identifiers:
urn: urn:nbn:ch:bel-epfl-thesis5071-9

Note: The status of this file is: EPFL only

 Record created 2011-04-27, last modified 2018-03-17

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