However, at 25 ☌, the moduli of gelatin-collagen composite hydrogel increased with the extension of time, its G′ increased about 18 times within 8 h, and the ratio of elastic modulus to viscous modulus (G″) increased 4.6 times, showing a significant aging effect of structural strength. The moduli of the assembled gel at 35 ☌ were equivalent to that of pure bovine tendon collagen system moreover, the system moduli didn’t change with time with elastic moduli (G′) of about 40 Pa. Studies on gel properties demonstrated that gelatin-collagen mixed solution had collagen-like assembly characteristics and assembly kinetics. Amino acid composition analysis test indicated that the content of polar amino acids and the sum of acidic and base amino acids for gelatin were higher than that of BPSC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) test results showed that the bovine tendon collagen had typical type-I collagen structural characterizations with two α chains of about 100 kDa and one β chain of about 200 kDa while the SDS-PAGE pattern of gelatin displayed bands continuously distributed from 30 to 200 kDa. The assembly properties and gel properties of this composite material were further studied. In this study, gelatin was ground into powders and swelled in neutral bovine tendon pepsin-soluble collagen solution (BPSC) to form a homogeneous gelatin-collagen mixture, in light of the swelling characteristics of gelatin in cold water. However, a low temperature (2–10 ☌) is required to prepare and store collagen solution, and neutral collagen solution denatures quickly above the room temperature. This is because gelatin and collagen have different soluble temperatures-gelatin is soluble in hot water (≥30 ☌) and swells in cold water. As the degradation product of collagen, gelatin is cheap, degradable and biocompatible, but few literatures have reported the research about gelatin-collagen composite materials. Pure collagen materials are expensive with poor mechanical properties, which need modifications in most cases.
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The refolded collagen still had weak self-assembly ability and formed a unique network-like structure containing small interlaced and closely combined fibers, which shows favorable cell compatibility and potential applications. In conclusion, the reconstruction of the a chains did not perfectly occur in a “head-to-head, tail-to-tail” manner in refolded collagen, as each a chain was participating in the reconstruction of multiple triple helix domains. The telopeptide did not significantly promote triple helix reconstruction.
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We found that the denatured collagen products from different sources (grass carp skin, bovine tendon) all showed a reconstruction of the triple helix conformation up to 60–75% of the value of natural collagen during the refolding process. Next, we observed the refolding behavior of the denatured collagen by removing urea through dialysis. The denaturation treatment severely destroyed the triple helix conformation of collagen, but had no significant effect on the primary structure of its a chains or the covalent cross-linking between a chains. In this study, using urea as a denaturant, we prepared a denatured natural collagen product and analyzed its structural changes. Exploration of the denaturation and refolding of natural collagen is important for the application of collagen and its denatured products.