The influence of tissue engineering procedure on collagen cross-links.
(2024) European Society of Tissue Regeneration in Orthopaedics and Traumatology (ESTROT) congress — Location: Helsinki (Finland) (10.June.2024)
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- Authors
Manon, Julie 
- et. al.
- Abstract
- Tissue engineering techniques can influence fundamental properties of native tissues, such as the intermolecular crosslinks within collagen triple helices, essential for maintaining tissue structure and function. The study assessed crosslink persistence in extracellular matrix (E CM after tissue engineering processes (decellularization, sterilization) using morphological and molecular analyses on native and treated human fascia lata (HFL). HFLs were collected from three deceased donors and decellularized following five chemical protocols , either with or without detergents (referred to as D1 D4 and D5, respectively). Additionally, γ irradiation (25kGy) was applied to investigate the i nfluence of sterilization on the arrangement of the ECM. The distribution of thick and thin collagen fibers was examined using polarized light microscopy ( on Sirius red stained sections. Furthermore, molecular analyses were conducted utili z ing Raman Spectros copy to examine tissue hydration and hydroxyproline content, as well as enzymatic and non enzymatic cross links. ELISA was also employed to assess the preservation of decorin (DCN), a crucial small leucine rich proteoglycan involved in fibrillogenesis. Of the five decellularization approaches tested, the fift h protocol (D5) showed a propensity to intensify tissue disorganization. This phenomenon was initially observed morphologically through PLM, where there was an increase in the proportion of thin fibers (+53.7%) and a decrease in the proportion of thick on es ( 32.6%). This observation was further supported by Raman spectroscopy analysis, which revealed a decrease in the amount of collagen enzymatic cross links ( 25.2%) and also by the significant decre ase of DCN ( 62.95%, p = 0.036) This approach also resulted in a statistically significant elevation of the tissue hydration, shown by the three biomarkers associated with collagen bound water (Intensity Ratio (IR) 3220/2943, p=0.023; IR 3333/2943, p=0.037; and IR 3457/2943, p=0.023 ). Radiation exposure of D5 HFL samples intensified the decrease in enzymatic cross links ( 29%, p = 0.037) in compar ison to native ones. However, the comparison with decellularized non sterilized samples did not yield statistic ally significant differences. The tissue hydration increased after decellularization and decreased after irradiation, without returning to basal levels. Overall, these results imply that the decellularization processes did not induce significant alterations in the collagen cross links, except for the D5 protocol, which significantly reduced the DCN level. Furthermore, the addition of γ irradiation resulted in outcomes that were not significantly different from those of decellularized non irradiated samples, although the reduction of enzymatic cross links became significant for D5 irradiated samples when compared to native tissues. These results could be further correlated with the mechanical properties of these tissues. Moreover, high performance liquid chromatography could be employed to achieve a more precise quantitative assessment of collagen cross links.
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Citations
Vettese, J., Manon, J., Chretien, A., Evrard, R., Fievé, L., Schubert, T., Lengelé, B., Behets Wydemans, C., Cornu, O., & et al. (2024). The influence of tissue engineering procedure on collagen cross-links. European Society of Tissue Regeneration in Orthopaedics and Traumatology (ESTROT) congress, Helsinki (Finland).