Intracellular Pathways Involved in Bone Regeneration Triggered by Recombinant Silk-silica Chimeras.

Biomineralization at the organic-inorganic interface is critical to many biology material functions in vitro and in vivo. Recombinant silk-silica fusion peptides are organic-inorganic hybrid material systems that can be effectively used to study and control biologically-mediated mineralization due to the genetic basis of sequence control. However, to date, the mechanisms by which these functionalized silk-silica proteins trigger the differentiation of human mesenchymal stem cells (hMSCs) to osteoblasts remain unknown. To address this challenge, we analyzed silk-silica surfaces for silica-hMSC receptor binding and activation, and the intracellular pathways involved in the induction of osteogenesis on these bioengineered biomaterials. The induction of gene expression of αVß3 integrin, all three Mitogen-activated Protein Kinsases (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. This induction of key markers of osteogenesis correlated with the content of silica on the materials. Moreover, computational simulations were performed for silk/silica-integrin binding which showed activation of αVß3 integrin in contact with silica. This integrated computational and experimental approach provides insight into interactions that regulate osteogenesis towards more efficient biomaterial designs.

A robust spectroscopic method for the determination of protein conformational composition - Application to the annealing of silk.

The physical and mechanical properties of structural proteins such as silk fibroin can be modified by controlled conformational change, which is regularly monitored by Fourier transform infrared spectroscopy by peak fitting of the amide I band envelope. Although many variables affecting peak shape are well established, there is no fixed methodology to compare and follow secondary structural differences without significant operator input especially where low frequency spectral noise is a problem. The aim of this contribution is to establish a method for such analyses to be carried at high levels of autonomy to prevent subjective or erroneous fitting. A range of approaches was trialled with optimal peak parameters selected based on overall goodness of fit and reproducibility of fit of replicate sample spectra. The method was successfully tested against reference proteins having contrasting ß content and the rationale for parameter selection is presented. Further, we applied this method to measure the effect of conformational change on the energy of the amide I band of silk fibroin during annealing. Energy changes were ca. 400 kJ mol-1 of fibroin. To confirm that this energy change was a consequence of increased hydrogen bonding we used a Thioflavin T staining method typically used to identify ß aggregate type structures in amyloid plaques. We propose that the approach described herein can aid in the development of silk based materials for biomedical applications where tuning of the physical and mechanical properties of the silk are needed to guarantee optimum activity. STATEMENT OF SIGNIFICANCE: The physical and mechanical properties of proteins including silk fibroin can be modified by controlled structural change, which is regularly monitored by Fourier transform infrared spectroscopy (FTIR) by peak fitting of the amide I band. Currently there is no fixed methodology to compare and follow secondary structural differences without significant operator input leading to subjectivity and error. This contribution establishes a method for such analyses to be carried at high levels of autonomy applicable to a wide range of proteins and the conformational changes have been quantified as a single energy change output, which clearly shows the progression of the annealing process used. We propose that the approach can help in the development of silk based materials for biomedical applications where tuning of the physical and mechanical properties of the silk are needed to guarantee optimum activity.

Quantifying the efficiency of Hydroxyapatite Mineralising Peptides.

We present a non-destructive analytical calibration tool to allow quantitative assessment of individual calcium phosphates such as hydroxyapatite (HAP) from mixtures including brushite. Many experimental approaches are used to evaluate the mineralising capabilities of biomolecules including peptides. However, it is difficult to quantitatively compare the efficacy of peptides in the promotion of mineralisation when inseparable mixtures of different minerals are produced. To address this challenge, a series of hydroxyapatite and brushite mixtures were produced as a percent/weight (0-100%) from pure components and multiple (N = 10) XRD patterns were collected for each mixture. A linear relationship between the ratio of selected peak heights and the molar ratio was found. Using this method, the mineralising capabilities of three known hydroxyapatite binding peptides, CaP(S) STLPIPHEFSRE, CaP(V) VTKHLNQISQSY and CaP(H) SVSVGMKPSPRP, was compared. All three directed mineralisation towards hydroxyapatite in a peptide concentration dependent manner. CaP(V) was most effective at inducing hydroxyapatite formation at higher reagent levels (Ca2+ = 200 mM), as also seen with peptide-silk chimeric materials, whereas CaP(S) was most effective when lower concentrations of calcium (20 mM) and phosphate were used. The approach can be extended to investigate HAP mineralisation in the presence of any number of mineralisation promoters or inhibitors.

Osteoinductive recombinant silk fusion proteins for bone regeneration.

Protein polymers provide a unique opportunity for tunable designs of material systems due to the genetic basis of sequence control. To address the challenge of biomineralization interfaces with protein based materials, we genetically engineered spider silks to design organic-inorganic hybrid systems. The spider silk inspired domain (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT)15 served as an organic scaffold to control material stability and to allow multiple modes of processing, whereas the hydroxyapatite binding domain VTKHLNQISQSY (VTK), provided control over osteogenesis. The VTK domain was fused either to the N-, C- or both terminals of the spider silk domain to understand the effect of position on material properties and mineralization. The addition of the VTK domain to silk did not affect the physical properties of the silk recombinant constructs, but it had a critical role in the induction of biomineralization. When the VTK domain was placed on both the C- and N-termini the formation of crystalline hydroxyapatite was significantly increased. In addition, all of the recombinant proteins in film format supported the growth and proliferation of human mesenchymal stem cells (hMSCs). Importantly, the presence of the VTK domain enhanced osteoinductive properties up to 3-fold compared to the control (silk alone without VTK). Therefore, silk-VTK fusion proteins have been shown suitable for mineralization and functionalization for specific biomedical applications. STATEMENT OF SIGNIFICANCE: Organic-inorganic interfaces are integral to biomaterial functions in many areas of repair and regeneration. Several protein polymers have been investigated for this purpose. Despite their success the limited options to fine-tune their material properties, degradation patterns and functionalize them for each specific biomedical application limits their application. Various studies have shown that the biological performance of such proteins can be improved by genetic engineering. The present study provides data relating protein design parameters and functional outcome quantified by biomineralization and human mesenchymal stem cell differentiation. As such, it helps the design of osteoinductive recombinant biomaterials for bone regeneration.

Influence of silk-silica fusion protein design on silica condensation in vitro and cellular calcification.

Biomaterial design via genetic engineering can be utilized for the rational functionalization of proteins to promote biomaterial integration and tissue regeneration. Spider silk has been extensively studied for its biocompatibility, biodegradability and extraordinary material properties. As a protein-based biomaterial, recombinant DNA derived derivatives of spider silks have been modified with biomineralization domains which lead to silica deposition and potentially accelerated bone regeneration. However, the influence of the location of the R5 (SSKKSGSYSGSKGSKRRIL) silicifying domain fused with the spider silk protein sequence on the biosilicification process remains to be determined. Here we designed two silk-R5 fusion proteins that differed in the location of the R5 peptide, C- vs. N-terminus, where the spider silk domain consisted of a 15mer repeat of a 33 amino acid consensus sequence of the major ampullate dragline Spidroin 1 from Nephila clavipes (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT). The chemical, physical and silica deposition properties of these recombinant proteins were assessed and compared to a silk 15mer control without the R5 present. The location of the R5 peptide did not have a significant effect on wettability and surface energies, while the C-terminal location of the R5 promoted more controlled silica precipitation, suggesting differences in protein folding and possibly different access to charged amino acids that drive the silicification process. Further, cell compatibility in vitro, as well as the ability to promote human bone marrow derived mesenchymal stem cell (hMSC) differentiation were demonstrated for both variants of the fusion proteins.