The influence of specific binding of collagen–silk chimeras to silk biomaterials on hMSC behavior

Abstract

Collagen-like proteins in the bacteria Streptococcus pyogenes adopt a triple-helix structure with a thermal stability similar to that of animal collagens, can be expressed in high yield in E. coli and can be easily modified through molecular biology techniques. However, potential applications for such recombinant collagens are limited by their lack of higher order structure to achieve the physical properties needed for most biomaterials. To overcome this problem, the S. pyrogenes collagen domain was fused to a repetitive Bombyx mori silk consensus sequence, as a strategy to direct specific non-covalent binding onto solid silk materials whose superior stability, mechanical and material properties have been previously established. This approach resulted in the successful binding of these new collagen-silk chimeric proteins to silk films and porous scaffolds, and the binding affinity could be controlled by varying the number of repeats in the silk sequence. To explore the potential of collagen-silk chimera for regulating biological activity, integrin (Int) and fibronectin (Fn) binding sequences from mammalian collagens were introduced into the bacterial collagen domain. The attachment of bioactive collagen-silk chimeras to solid silk biomaterials promoted hMSC spreading and proliferation substantially in comparison to the controls. The ability to combine the biomaterial features of silk with the biological activities of collagen allowed more rapid cell interactions with silk-based biomaterials, improved regulation of stem cell growth and differentiation, as well as the formation of artificial extracellular matrices useful for tissue engineering applications.

Definition of the Native and Denatured Type II Collagen Binding Site for Fibronectin Using a Recombinant Collagen System

“…Previously, the potential to obtain collagenous proteins with controllable biological activity modules using the bacterial collagen system has been demonstrated, leading to improved human mesenchymal stem cell growth in two-and three-dimensional cultures (24). To further demonstrate SCl-Fn as a suitable bioactive material, human megakaryocytes were cultured on SCl-Fn in the presence of Fn.…”

“…1). The human collagen sequences were inserted between two tandem CL domains of bacterial collagen because this design was previously reported to be successful for conferring biological activity (24,29). Recent studies by Erat et al (15) indicated a peptide with residues 778 -799 from the ␣1 chain of type I collagen bound tightly to Fn.…”

“…Here, a recombinant bacterial collagen-like protein system is employed to characterize the amino acid sequences in native and denatured collagen required for Fn binding. A collagen-like protein found in Streptococcus pyogenes, which is readily expressed and easily modified in Escherichia coli, was previously shown to have a triple helix structure and stability similar to that of human fibrillar collagens, even though post-translationally formed hydroxyproline (Hyp) is absent (22)(23)(24). Chimeric collagen-like proteins were generated by inserting potential Fn binding sequences from human collagen type II between bacterial collagen modules.…”

Bioengineered collagens

“…For example, integrin and heparin binding domains have been replaced into the S. pyogenes CL domain sequence, 37,38 while an integrin site and a fibronectin site have each been inserted as extra residues into the CL domain. 39 In addition, the collagenase cleavage in type III collagen has been examined when inserted between two CL domains. 40 The concept of producing dimers, or small oligomers of the bacterial collagens, allows larger molecules to be produced-for example, similar in size to mammalian fibrillar collagens-but fermentation yields would be expected to be lower.…”

“…Recently, the addition of a silk-like motif, (GAGAGS) n , to the C-terminal of the S. pyogenes CL domain has been shown to enable binding to a silk substrate, providing the opportunity to develop more complex composite structures. 39 A key biotechnology consideration is what products could be developed from the new bacterial collagen-based materials? There would certainly seem to be opportunities in biomedical products, as with mammalian collagens, either where the new collagens are used as coatings or fabricated into a specific device, to provide enhanced products for specific applications.…”