# Routes towards Novel Collagen-Like Biomaterials
## 2 Collagen as a Biomaterial
### Collagen-Analogues as Basis for Future Biomaterials  
As mentioned above, biomaterials based on natural collagens today are still mostly decellularized animal tissues that have been mechanically or chemically processed into the desired morphologies. This approach carries the intrinsic risk of adverse immunological reactions in the receiving patient due to allergies towards the graft [33], as well as the spreading of viral or prion-based contaminants [34]. The currently used methods for the extraction of soluble collagen from tissue include the use of mild solvents (phosphate buffered salt solution; dilute acetic acid) [9], which yield full length triple helices that still contain the terminal cltopeptides. The best yields are gained when collagen is extracted from young tissues, such as calf skin and rat tail. Another option employs proteases such as pepsin, which utilizes the enzyme's unspecific activity to digest all non-collagen-like domains and therefore removes telopeptides, which results in higher yields even from more strongly cross-linked tissues [35]. Recently, collagen-based scaffolds for tissue engineering have been produced from solubilized and purified collagen that often has been blended with other polymers due to the limited availability of the soluble collagen source material [36; 37]. When purified collagen is required for the production of mechanically stable biomaterials such as grafts for tendon replacement, the trade-off between batch-to-batch homogeneity and yield will always be a restricting factor. While large amounts of animal tissue are available for the extraction of collagen not intended for clinical use, the current sources of human collagen are limited to scarcely available extractable tissues (such as dermis) [8] and biotechnology produced collagen made from human fibroblast cell culture (e.g., CosmoDerm) [38]. To overcome these limitations, different approaches have been made to find substitutes for natural vertebrate collagen which have adequate properties for tissue engineering, regenerative medicine and other biomedical applications. Collagen mimetic peptides were originally used in determining the structure, stability and folding kinetics of collagen and collagen-like sequences [39; 40; 41], which provided remarkable insight into the role of each amino acid within the (GXY)\({}_{n}\)-repetitive sequence and led to attempts at stabilizing short triplehelical peptides by incorporating non-canonical amino acids, such as 4-fluoroproline [42] or N-isobutylglycine [43]. Furthermore, these experiments identified the reason for the comparably slow folding of nucleated triplehelices to be based upon the slow cis/trans-isomerization rates of residues in the X- and Y-positions [44; 45; 46]. Using these peptides, researchers also noted that the formation of gelatin-like networks could be largely avoided by incorporating amino- or carboxyterminal nucleation sites that would  
Figure 1: (**a**) Collagen microfibers are produced within a PDMS microfluidic chip from a soluble precursor solution (pH 3 in dilute acetic acid). Thread formation takes place at the junction that brings the spinning solution in contact with the pH 8 buffer solution. The flowrate of each solution is tightly controlled by independent syringe pumps; (**b**) the resulting collagen fiber is highly homogeneous and has remarkable mechanical properties comparable to that of tendon, while still being flexible enough for downstream processing. Modified with permission from [11] (ACS, 2016). promote helix formation within the cross-linked trimer at a much higher rate than between non-nucleated single-chain constructs [44; 46]. Possible nucleation sites can be TRIS-scaffolds [47; 48], 1,2,3-propan-tricarboxylic-acid scaffolds [49], collagen III derived cystine knots [50] and bacterial or bacteriophage-derived protein domains [20; 51]. All of these approaches use peptide synthesis, which is typically limited to a maximal peptide length of 30-50 amino acids.