Testing the utility of rationally engineered recombinant collagen-like proteins for applications in tissue engineering.

Collagens are attractive proteins as materials for tissue engineering. Over the last decade, significant progress has been made in developing technologies for large-scale production of native-like human recombinant collagens. Yet, the rational design of customized collagen-like proteins for smart biomaterials to enhance the quality of engineered tissues has not been explored. We mapped the D4 domain of human collagen II as most critical for supporting migration of chondrocytes and used this information to genetically engineer a collagen-like protein consisting of tandem repeats of the D4 domain (mD4 collagen). This novel collagen has been utilized to fabricate a scaffold for support of chondrocytes. We determined superior qualities of cartilaginous constructs created by chondrocytes cultured in scaffolds containing the mD4 collagen in comparison to those formed by chondrocytes cultured in bare scaffolds or those coated with wild-type collagen II. Our results are a first attempt to rationally engineer collagen-like proteins with characteristics tailored for specific needs of cartilage engineering and provide a basis for rational engineering of similar proteins for a variety of biomedical applications.

Guilty by association: some collagen II mutants alter the formation of ECM as a result of atypical interaction with fibronectin.

Among the structural components of extracellular matrices (ECM) fibrillar collagens play a critical role, and single amino acid substitutions in these proteins lead to pathological changes in tissues in which they are expressed. Employing a biologically relevant experimental model consisting of cells expressing R75C, R519C, R789C, and G853E procollagen II mutants, we found that the R789C mutation causing a decrease in the thermostability of collagen not only alters individual collagen molecules and collagen fibrils, but also has a negative impact on fibronectin. We propose that thermolabile collagen molecules are able to bind to fibronectin, thereby altering intracellular and extracellular processes in which fibronectin takes part, and we postulate that such an atypical interaction could change the architecture of the ECM of affected tissues in patients harboring mutations in genes encoding fibrillar collagens.

Position of single amino acid substitutions in the collagen triple helix determines their effect on structure of collagen fibrils.

Collagen II fibrils are a critical structural component of the extracellular matrix of cartilage providing the tissue with its unique biomechanical properties. The self-assembly of collagen molecules into fibrils is a spontaneous process that depends on site-specific binding between specific domains belonging to interacting molecules. These interactions can be altered by mutations in the COL2A1 gene found in patients with a variety of heritable cartilage disorders known as chondrodysplasias. Employing recombinant procollagen II, we studied the effects of R75C or R789C mutations on fibril formation. We determined that both R75C and R789C mutants were incorporated into collagen assemblies. The effects of the R75C and R789C substitutions on fibril formation differed significantly. The R75C substitution located in the thermolabile region of collagen II had no major effect on the fibril formation process or the morphology of fibrils. In contrast, the R789C substitution located in the thermostable region of collagen II caused profound changes in the morphology of collagen assemblies. These results provide a basis for identifying pathways leading from single amino acid substitutions in collagen II to changes in the structure of individual fibrils and in the organization of collagenous matrices.