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. 2019 Aug 26:7:203.
doi: 10.3389/fbioe.2019.00203. eCollection 2019.

Investigations of Processing-Induced Structural Changes in Horse Type-I Collagen at Sub and Supramolecular Levels

Alberta Terzi  1 , Nunzia Gallo  2 , Simona Bettini  2 , Teresa Sibillano  1 , Davide Altamura  1 , Lorena Campa  3 , Maria Lucia Natali  3 , Luca Salvatore  2 , Marta Madaghiele  2 , Liberato De Caro  1 , Ludovico Valli  4 , Alessandro Sannino  2 , Cinzia Giannini  1
Affiliations

Investigations of Processing-Induced Structural Changes in Horse Type-I Collagen at Sub and Supramolecular Levels

Alberta Terzi et al. Front Bioeng Biotechnol. .

Abstract

The aim of this work is to evaluate the effects of different extraction and material processing protocols on the collagen structure and hierarchical organization of equine tendons. Wide and Small Angle X-ray Scattering investigations on raw powders and thin films revealed that not only the extraction and purification treatments, but also the processing conditions may affect the extent of the protein crystalline domain and induce a nanoscale "shield effect." This is due to the supramolecular fiber organization, which protects the atomic scale structure from the modifications that occur during fabrication protocols. Moreover, X-ray analyses and Fourier Transform Infrared spectroscopy performed on the biomaterial sheds light on the relationship between processing conditions, triple helical content and the organization in atomic and nanoscale domains. It was found that the mechanical homogenization of the slurry in acidic solution is a treatment that ensures a high content of super-organization of collagen into triple helices and a lower crystalline domain in the material. Finally, mechanical tensile tests were carried out, proving that the acidic solution is the condition which most enhances both mechanical stiffness and supramolecular fiber organization of the films.

Keywords: FT-IR; X-rays; biomaterial; medical devices; stiffness; structural modification; type I collagen.

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Figures

Figure 1
Figure 1
SDS PAGE of collagen suspensions in comparison with the marker M (broad range 37–250 kDa), CC was used as the control for the purity assessment of TYP1CH and TYP1EN.
Figure 2
Figure 2
(A) Diffracted intensity distribution of TYP1CH_OMO_A; (B) Corresponding 1D WAXS profile. (C) Gaussian fit elaboration and FWHM calculation of the equatorial diffraction peak.
Figure 3
Figure 3
2D WAXS diffraction patterns of A type raw collagen samples and thin films fabricated by homogenization protocol (OMO) and with crosslinking treatment (OMO_DHT).
Figure 4
Figure 4
1D SAXS profile of TYP1CH_OMO_A (A). Gaussian fit elaboration and FWHM calculation of the 6th diffraction peak (B).
Figure 5
Figure 5
1D SAXS diffraction profiles of A type raw collagen samples and thin films fabricated by homogenization protocol (OMO) and with crosslinking treatment (OMO_DHT).
Figure 6
Figure 6
(A) FT-IR spectra in the 3,600–700 cm−1 range of the raw equine collagens: CC (black), TYP1EN (dark cyan) and TYP1CH (blue); (B) Amide I peak de-convolution of α-helix (green line) and triple helix contributes (gray line) elaborated by experimental curve (scatter plot) in CC raw equine collagens.
Figure 7
Figure 7
Amide I peak de-convolution of α-helix (green line) and triple helix contributes (gray line) elaborated by experimental curve (scatter plot) in CC collagen samples: not crosslinked (A) and DHT (B).
Figure 8
Figure 8
(A) Young's moduli of equine collagen-based DHT treated films produced by TYP1CH, TYP1EN and CC isoforms. Histogram directly compares the average moduli for each sample as a function of starting material, crosslinking treatment and processing. Error bars represent the standard deviation. (B) 1D SAXS profiles of TYP1CH DHT films obtained from hydrated (collagen B) and lyophilized (collagen A) collagen and treated by homogenization (OMO) and acidic dissolution (AA).
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