Collagen characterization in two types of matrices: osteogenesis imperfecta mouse tendon and human fascia lata tissue engineering

The extracellular matrix (ECM) is a three-dimensional scaffold that provides structural and mechanical support to tissues. Collagen is the main macromolecule of this network with its type I being the most widely expressed. This heterotrimer undergoes several physiological post-translational modifications (PTM). The analysis of these PTM and their potential alteration inform on the structural and mechanical properties of tissues. In this project, ECM was analyzed in osteogenesis imperfecta mouse (oim) tendons as well as in tissue engineering of human fascia lata (HFL) and human periosteum (HP). The first objective was to compare tendons of oim and wild type (WT) adult mice. Density and birefringence were significantly lower in oim than WT tendons (p < 0.005 and p = 0.001, respectively) attesting a difference in collagen bundle organization. Their molecular composition, assessed by Raman spectroscopy, was characterized by a significantly higher proportion of pentosidine (p < 0.0001) and hydroxyproline (p < 0.05) than that of WT tendons. This method also highlighted a lower proportion of bound water in oim ones. This was associated with a significant lower tendon mass assessed by sequential dehydration process (p < 0.0001). Mechanical tests showed a significantly lower elastic modulus (p < 0.05), ultimate stress (p < 0.05) and stress relaxation (p < 0.05) in oim than WT tendon. In the second part of this work, HFL and HP were harvested from cadaveric donors (n = 3) and decellularized following five different chemical protocols with/without soap (D1-D4 and D5, respectively). HFL tissues were also gamma-irradiated (minimum 25kGy) in order to study the impact of sterilization on the ECM organization. Picro-sirius red stained sections of non-irradiated HFL and HP were analyzed under polarized light microscopy (PLM) and highlighted that the D5 protocol tended to further disorganize HFL, as more thin fibers (+53.7%) and less thick ones (-32.6%) were visible. Raman spectroscopy showed less collagen enzymatic cross-links (-25.2%, p = 0.19) in the D5 HFL samples, as well as statistically increased collagen-bound water, as highlighted by the corresponding biomarkers (Intensity Ratio (IR) 3220/2943, p = 0.023; IR 3333/2943, p = 0.037 and IR 3457/2943, p = 0.023). However, the D1 protocol tended to disorganize the HP more than the other ones but without any statistical difference. D1 tissues recorded more thin fibers (+189%) and less thick ones (-68.24%) but also less enzymatic cross-links (-37.35%). Irradiation increased the loss of enzymatic cross-links proportion in D5-HFL samples, which became statistically different from the native ones (-29.4%, p = 0.037). However, there was no statistical difference between the decellularized tissues before and after irradiation for any of the Raman measured parameters. In conclusion, our data showed a relationship between collagen structure and composition. Further progress in the characterization of oim tendons will open new treatment possibilities. Similarly, future clinical applications of decellularized matrices can be envisaged due to their minimal alteration.