RGD-Functionalized Bioengineered Spider Dragline Silk Biomaterial
Bini, E., Foo, C.W.P., Huang, J., Karageorgiou, V., Kitchel, B., and Kaplan, D.L.Biomacromolecules, 7, 11, 3139 - 3145, 2006, 10.1021/bm0607877
Summary: This paper details an experiment done with recombinant spider silk dragline proteins in an effort to produce a viable tissue regeneration scaffold. The spider silk was modified to include RGD domains that promote cell binding. This particular experiment tested human bone marrow mesenchymal stem cells (hMSCs) and their tendency to differentiate into bone cells on recombinant spider silk (both displaying and not displaying RGD domains) versus tissue culture plastic.
The basic repeating sequence of amino acids was derived from analysis of the silk of Nephila clavipes. Proteins expressing only this sequence were produced from bacteria, as well as proteins with a modified sequence to include the RGD domain. In either case, the protein was 15 monomeric units long. The protein produced was quantified using SDS-PAGE gels, BCA assays, and Western blots, along with other assays.
The hMSCs were seeded on 3 different surfaces for the experiment: a film of the modified protein with RGD domains, a film of the protein without RGD domains, and sterile polystyrene. They were all grown in the same media, and cell response was determined through colorimetric assays measuring calcium content. The authors determined that both protein films resulted in a higher calcium content than tissue culture plastic, but the protein modified to display RGD domains did not produce a greater response than the protein without the RGD domains. The calcium content was actually higher on the protein without RGD domains, indicating that the native form of the protein is enough to allow cells to attach.
Significance: Spider silk has generated a fair amount of interest as a potential tissue regeneration scaffold material. In addition to its desirable mechanical properties, it is a protein polymer, which can be easily modified to introduce new functions, such as the RGD binding domains tested in the above experiment. The experiment shows that cells attach and differentiate better on spider silk-based films than on tissue culture plastic. While this is not completely conclusive about spider silk's viability as a scaffold material, it shows some promise as a material that can be used to aid cell differentiation and tissue formation.
8 comments:
I understand that the spider dragline silk is naturally a very strong material, but is not easy to mass produce. Using E. Coli to generate the silk, were there any tests done to confirm that the silk produced still kept its unique mechanical properties?
Also, were any in vivo tests performed to check the biocompatability of the silk in an organism compared to just the cell culture? You mention that the cells produced more calcium on the silk scaffold, were any other tests done to check that this meant that the scaffold was inducing bone mineralization?
Has the spider silk scaffold only been tested with human bone marrow mesenchymal stem cells (hMSCs) so far, or is there other research going on with different cell types? Could it be possible that different cell types would attach and act differently when grown on this scaffold?
Jeff:
Studies have been done about the mechanical properties of engineered spider silks. The actual strength depends on how you process the silk and what kind of material you make from it (fibers, films, mats), but at the very least, properly processed engineered silk is almost as good as native silk. The authors of this paper were less interested in studying mechanical properties, though, and they assume the mechanical properties will be good enough for a scaffold.
This particular paper did not discuss any in vivo tests with spider silk. It focused a good deal on characterizing the chemistry and structure of the manufactured protein film, since their purpose was mainly to see he effect of RGD-functionalizing the protein. As such, they didn't do any test other than calcium production, since they weren't interested as much in inducing osteogenic outcomes as they were comparing the various material types (RGD silk, silk, and polystyrene).
Dana:
This particular paper only uses hMSCs. There probably have been studies using other cell types, but not necessarily with RGD-functionalized silk. I don't know what papers specifically have tested other cell types, because that's beyond the scope of this assignment. Sorry.
The RGD sequence is very generic sequence for integrin binding. So, any cell in the vicinity will bind to the RGD codon. I know this paper only talked about an in vitro experiment and didn't do any in vivo experiments. But did it say anything about implanting this silk scaffold into the body? It might have problems with biofouling and other cell adhesion.
Hey Eric,
Interesting article you have here. To go off some of the other comments you've received in regards to biocompatibility, I was curious about whether any considerations have been made in regards to the spider silk scaffold degrading over time. If the silk scaffold could degrade over time then do you think it would be possible to implant the newly grown bone cells into a body for an in vivo test?
Why do you think the non-RGD silk actually produced better attachment compared with the RGD silk? RGD is suppose to help attachment; what would make it produce a contrary effect in this case?
Eric:
Can you clarify for me how these spider silk scaffold are generated from the 15 monomer proteins? Is there some sort of biological reaction involved to generate them mesh?
Also, do you think this biomaterial will be feasible for large-scale research or will this be limited to a small laboratory setting?
Lavanya:
This paper wasn't really concerned with in vivo tests. However, there have been some in vivo studies done, and I believe that spider silk has generally been found to induce only a mild immune response. As for unwanted cell attachment due to the nonspecificity of the RGD binding domain, that might not be an issue. The scaffold is intended to be planted in a very specific location in the body, so any cells that attach would presumably be desired. I can't say for sure, though.
Robbedsmiler:
The degradation of spider silk has been studied, but not in this paper. It is definitely a design parameter you'd want to take into account for an implant, and is to some degree controllable. Ideally the scaffold would only persist for as long as it takes for the cells to grow their own ECM, so after several months the scaffold should be gone. Silk has the advantage of being resorbable, meaning the products of degradation can be dealt with by natural processes in the body.
Tue:
The authors suggested that the natural structure of spider silk protein polymers is enough to induce cell binding, such that adding an RGD domain would have a minor effect. Adding the RGD to the primary peptide sequence may have caused a change in the secondary/tertiary structures of the polymer, making it more difficult for cells to bind. This effect may have also caused the RGD domains to be unavailable for binding.
Aron:
The processing into a mesh just involved placing the recombinant proteins into a solution of hexafluoroisopropanol, pipetting the solution onto ZnS crystals, and allowing it to air dry and create a film. The authors would then use a methanol treatment to make the film insoluble, then either use the film directly or process it into fibers and make a mesh.
The processing probably wont be difficult to scale up to a mass-production level, but there is still the issue of mass-producing the protein.
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