Abstract
Recombinant spider silk has the potential to provide a new generation of biomaterial scaffolds as a result of its degree of biocompatibility and lack of immunogenicity. These recombinant biomaterials are, however, reported to exhibit poor cellular adhesion which limits their potential for use in applications such as tissue engineering and regenerative medicine. In this study, a simple chemical functionalization approach is described that specifically addresses this issue and significantly improves the adhesion of human mesenchymal stem cells (CiMSCs)to a recombinant spider silk biomaterial. Thisutilizes copper-catalyzed or strain-promoted azide-alkyne cycloaddition (CuAAC/SPAAC) "click" chemistry to covalently attachcyclo(RGDfK) peptides to the azide group of l-azidohomoalanine, a methionine analogue previously site specifically incorporated into the primary sequence of a thioredoxin (TRX)-tagged silk fusion protein, TRX-4RepCT, to give TRX3Aha -4RepCT3Aha . This method is used to producecyclo(RGDfK) functionalized films and macroscopic fibers. Over 24 h,cyclo(RGDfK) functionalized TRX3Aha -4RepCT3Aha films and 4RepCT3Aha fibers display significantly improved performance inCiMSCculture,yielding far greater cell numbers than the controls. Thisapproach circumvents the previously observedlack of cell adhesion, thus allowing spider silk derived biomaterials to be used where such adhesion is critical, in tissue engineering, regenerative medicine and wound healing.
Highlights
Recombinant spider silk has the potential to provide a new generation of interdisciplinary field of research that biomaterial scaffolds as a result of its degree of biocompatibility and lack of utilizes both man-made and natural polyimmunogenicity
Addresses this issue and significantly improves the adhesion of human. Both have been extensively studied and mesenchymal stem cells (CiMSCs) to a recombinant spider silk biomaterial. This utilizes copper-catalyzed or strain-promoted azide–alkyne cycloaddition (CuAAC/strain promoted azidealkyne cycloaddition (SPAAC)) “click” chemistry to covalently attach cyclo(RGDfK) peptides to the azide group of l-azidohomoalanine, a methionine anaprocessed into several different morphologies including gels and fibrous mats that support the culture of mammalian cells.[1]
We have previously demonstrated the incorporation of the bioorthogonal methionine analogue, l-azidohomoalanine (l-Aha), into the miniature dragline spidroin TRX-4RepCT using a methionine auxotrophic E. coli strain (DL41).[18]
Summary
Recombinant spider silk has the potential to provide a new generation of interdisciplinary field of research that biomaterial scaffolds as a result of its degree of biocompatibility and lack of utilizes both man-made and natural polyimmunogenicity. TRX3Aha-4RepCT3Aha fusion protein, and 4RepCT3Aha fibers were functionalized with cyclo(RGDfK) peptides, a known integrin ligand that improves cellular adhesion.[24] Functionalization was achieved using CuAAC (films only, yielding TRX-4RepCT-RGDCuAAC) and for the first time in recombinant spider silk research, copper-free strain promoted azide-alkyne cycloaddition (SPAAC) (films and fibers, yielding TRX-4RepCT-RGDSPAAC, and fiber-RGD respectively). Both techniques allowed simple and rapid functionalization of the silk material, SPAAC permitted functionalization. This could be used to produce 2D and 3D silk materials of increasing complexity that more closely mimic tissue specific niches
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