Zaira Martín-Moldes, Davoud Ebrahimi, Robyn Plowright, Nina Dinjaski, Carole C. Perry, Markus J. Buehler and David L. Kaplan
Department of Biomedical Engineering, Tufts University, Medford, MA, 02155, USA
Biomineralization at the organic-inorganic interface is critical to many biology material functions in vitro and in vivo. Bioengineered silks are attractive biomaterial substrates as they provide scaffolds for tissue regeneration due to biocompatibility, the ability to fine tune properties through sequence modification and processing, and the potential to engineer these proteins to incorporate diverse and selective functional domains. Specifically, recombinant silk-silica fusion peptides are organic-inorganic hybrid material systems that can be effectively used to study and control biologically-mediated mineralization. Yet the mechanisms by which these functionalized silk composites trigger the differentiation of human mesenchymal stem cells (hMSCs) to osteoblasts is unknown. High performance supercomputing simulations were utilized to synergistically identify relationships between the sequence design of silk protein-silica binding peptides and the effect on silicification. In addition, intracellular pathways involved in the process of mineralization when stem cells were grown on these silica substrates were also addressed.
In this integrated experimental-simulation approach, six silk sequence constructions were pursued, based on designs with three key domains: a core silk domain for materials assembly, a histidine tag for purification, and a silica binding peptide domain for silicification. The results showed that the addition of the silica and histidine domains reduced β-sheet structure in the recombinant protein materials, and increased solvent-accessible surface area to the positive charged amino acids, leading to higher levels of silica precipitation. But also, that binding of silica particles did not affect the protein structure of these samples. Moreover, the simulations showed that the location of the charged biomineralization domain had a minor effect on protein folding, in agreement with the experimental data. To determine key intracellular pathways involved in the induction of osteogenesis on these bioengineered biomaterials gene expression levels were monitored. The induction of gene expression of αVβ3 integrin, all three Mitogen-activated Protein Kinsase (MAPKs) as well as c-Jun, Runt-related Transcription Factor 2 (Runx2) and osteoblast marker genes was demonstrated upon growth of the hMSCs on the silk-silica materials. Cells growing on silk films without silica showed no induction of these genes. Furthermore, the induction of key markers of osteogenesis correlated with the content of silica on the materials. Additionally, computational simulations were performed for silk/silica-integrin binding which showed activation of αVβ3 integrin in contact with silica. The use of a blocking antibody against integrin αVβ3 showed an elimination of the induction of genes observed for the cells cultured on silk-silica films related to MAPK and TF, confirming involvement in osteogenesis.
This integrated modeling and experimental approach provides insight into sequence-structure-function relationships for control of mineralized protein biomaterial structures. This insight is critical for future design optimization of fusion proteins to generate mineralized biomaterials.
Funded: NIH U01 EB014976.