Casado Losada, Isabel (Department of Orthopedics and Trauma-Surgery, Division of Trauma-Surgery, Medical University of Vienna. Ludwig Boltzmann Institute for Traumatology, the Research Center in Cooperation with AUVA. Austrian Cluster for Tissue Regeneration. )


Cell infiltration is essential for the repopulation of dense materials in tissue engineering. During that process, several factors, such as scaffold topography, mechanical properties or porosity play a key role. These acquire special importance when designing the materials to substitute and regenerate articular cartilage. Because of their similar structure and composition, decellularized cartilage scaffolds represent an ideal candidate. Our group previously developed a biomaterial from auricular cartilage, which allowed us to study cell migration [1]. Removing the elastic fibres and depleting the glycosaminoglycans (GAGs) leaves a network of open channels. This process reduces the matrix density, altering their mechanical properties and enhances the elastic fibre removal process. In this study, we aimed to investigate the influence of GAG removal on the mechanical properties and cellular ingrowth on decellularized auricular cartilage scaffolds.
Scaffolds were harvested from bovine ears by punching 8mm diameter discs as described previously [1]. Briefly, scaffolds were cut to a standard thickness and subjected to several freezing-thawing cycles. Afterwards, an enzymatic treatment comprised of pepsin and elastase was used. Because pepsin is the enzyme that removes the GAGs, a concentration series from 0 to 1 mg/mL was used. Elastase, the enzyme that depletes the elastic fibres was maintained constant at a concentration of 0.03 U/mL. First, GAG content was quantified by histology (Alcian blue staining) and blyscan assay. Second, fluorescent-labelled adipose-derived stromal cells (ASCs) and human articular chondrocytes were seeded on the scaffolds and cultured under dynamic conditions for one week. Cell infiltration was monitored by confocal microscopy and immunohistochemistry. Furthermore, the mechanical properties were measured by a stepwise compressive test using a Zwick uniaxial testing machine. The viscosity and elasticity of the samples were further computed using a mathematical model. For comparison of two groups unpaired t-test or Mann–Whitney test was chosen according to distribution.
A concentration series of pepsin was used to modify the GAG content, which was inversely correlated. Residual GAG concentration after high and low pepsin concentrations differered significantly (1 mg/mL pepsin: 0.058 0.014 mg/µg vs. 0.2 mg/mL pepsin: 0.15 0.042 mg/µg; p = 0.0022). Cellular infiltration, however, followed the inverse trend. Higher concentrations of pepsin increased cellular infiltration. Nevertheless, even the lowest tested concentration (0.2 mg/mL) maintained adequate levels of cell migration into the open channels. Interestingly, cells were unable to penetrate the scaffolds treated without pepsin, forming a monolayer on the surface. In the slower infiltrating chondrocytes, the effect of matrix treatment was even more visible than for ASCs. Additionally, the mechanical properties followed a similar pattern. Native scaffolds and those treated with 0.2 mg/mL pepsin showed a viscoelastic behaviour and higher stiffness. On the contrary, concentrations above 0.4 mg/mL led to a more viscoplastic and fluid-like behaviour.
Auricular cartilage scaffolds are a suitable tool to study cell infiltration. Pepsin reduces considerably the GAG content, leading to reduced stiffness. Cell migration into the scaffolds is highly dependent on both parameters, which are crucial for scaffold development.
1. S. Nürnberger et al., Acta Biomater. 86, 207–222 (2019)"


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