(A-34) Collagen and Beyond: A Comprehensive Comparison of ECM Properties Derived from Different Human-Derived Sources for Tissue Engineering Applications

Introduction:: Collagen is a widely used biomaterial for tissue engineering and regenerative medicine applications, owing to its abundance in the human body [1]. Although collagen-based hydrogels are commonly employed in prior studies, use of single component systems do not recapitulate the physical and biological complexities of the native tissue ECM. Collagen, along with proteoglycans, glycosaminoglycans (GAGs), glycoproteins, and various growth factors, forms the extracellular matrix (ECM) that contributes to its complexity and diversity in different tissues [2]. Multicomponent ECM hydrogels derived from decellularized tissues have also been shown to support cell adhesion, proliferation, and differentiation, and can be beneficial for a range of different biomedical applications including 3D cell culture, organoids, and bioprinting [3,4]. The goal of this study was to compare the physicochemical and biological properties of ECM hydrogels derived from four different human tissues – skin, bone, fat and fetal. Pure collagen hydrogels were used as control. Physical characterization of tissue ECM hydrogels and assessment of cell response of cord mesenchymal stem cells (CMSCs) encapsulated in ECM hydrogels were performed to enable judicious selection of human-derived ECM for tissue-specific biomedical applications.


Materials and Methods:: ECM was extracted from different human-derived tissue sources by employing a proprietary decellularization and solubilization protocol. The resulting ECM was lyophilized prior to use in all the experiments. The efficacy of the decellularization process was evaluated by quantifying the remnant DNA using Quant-iT picogreen dsDNA kit. The purity of different tissue ECMs was characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-Page). To synthesize the tissue-specific ECM hydrogels, lyophilized ECM was solubilized in 15 mM HCl, mixed with 10x PBS, neutralized with 1M NaOH, and incubated at 37 °C for 60 min to induce fibrillogenesis. The polymerization rate of ECM solutions derived from different tissue sources was determined using a gelation kinetics assay. Hydroxyproline assay was used to quantify the amount of hydroxyproline (Hyp) in ECM solutions as a measure of the total collagen content. Alamar Blue assay was used to measure cell metabolic activity of CMSCs cultured on 2D ECM films and CMSCs encapsulated in 3D ECM hydrogels. Rheological properties of the ECM inks were measured using a Discover Series HR30 Rheometer. Drop-on-demand printing technique was used to assess the print fidelity of the ECM inks. Statistical analysis was performed using one-way with Tukey post hoc and statistical significance was set at p < 0.05.

Results, Conclusions, and Discussions:: SDS-page analysis showed the presence of γ, β, α1 and α2 collagen chains for all tissue ECMs (Figure 1A). DNA removal post decellularization was > 90% for all tissue ECM confirming that the decellularization protocols employed were efficacious (Figure 1B). Hyp quantification assay showed that collagen content in fetal ECM was comparable to the control (pure collagen) indicating that fetal ECM is predominantly composed of collagen (Figure 1C). However, collagen content in skin ECM, bone ECM and fat ECM were significantly lower (p < 0.05) than pure collagen and fetal ECM indicating the presence of other matrix proteins. Compositional variations in ECM derived from different tissue sources significantly impacted the gelation kinetics of the hydrogels with the collagen control hydrogels showing the fastest polymerization rate compared to ECM derived from different tissues suggesting that these ECM solutions compose of blends of collagen and other matrix proteins (Figure 2). Mechanical assessment via uniaxial compression testing revealed variations in the compressive modulus of different tissue ECM hydrogels, with skin ECM showing significantly higher modulus, and fetal ECM showing the lowest (Figure 3A, 3B). The stability of ECM hydrogels depended on the tissue source, with pure collagen hydrogels degrading the fastest and skin and fetal ECM hydrogels being more stable than bone and skin ECM hydrogels (Figure 3C). Differences between the tissue-specific ECM hydrogels in 3D hydrogels were observed, with the fetal ECM hydrogels exhibiting the highest metabolic activity at day 7 (Figure 4). Finally, rheological characterization revealed that all ECM-derived inks exhibited shear thinning properties and skin-derived ECM inks were most suitable for extrusion-based 3D bioprinting (Figure 5). Overall, results from this study demonstrate that the physicochemical and biological properties of ECM hydrogels vary significantly depending on the tissue source. Therefore, careful selection of tissue source is important for development of ECM-based constructs for tissue engineering and regenerative medicine therapies.


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References (Optional): : [1] Parenteau-Bareil, Rémi, et al, Materials 3.3 (2010):1863-1887. [2] Theocharis, Achilleas D., et al., Advanced drug delivery reviews (2016): 4-27. [3] Karamanos, Nikos K., et al., The FEBS journal 288.24 (2021):6850-6912. [4] Mollica, Peter A et al., Acta Biomaterialia (2019):201-213.