Impact of Endotoxins in Gelatine Hydrogels on Chondrogenic Differentiation and Inflammatory Cytokine Secretion In Vitro

Abstract

Gelatine methacryloyl (GelMA) hydrogels are widely used in studies aimed at cartilage regeneration. However, the endotoxin content of commercially available GelMAs and gelatines used in these studies is often overlooked, even though endotoxins may influence several cellular functions. Moreover, regulations for clinical use of biomaterials dictate a stringent endotoxin limit. We determined the endotoxin level of five different GelMAs and evaluated the effect on the chondrogenic differentiation of equine mesenchymal stromal cells (MSCs). Cartilage-like matrix production was evaluated by biochemical assays and immunohistochemistry. Furthermore, equine peripheral blood mononuclear cells (PBMCs) were cultured on the hydrogels for 24 h, followed by the assessment of tumour necrosis factor (TNF)-α and C–C motif chemokine ligand (CCL)2 as inflammatory markers. The GelMAs were found to have widely varying endotoxin content (two with >1000 EU/mL and three with <10 EU/mL), however, this was not a critical factor determining in vitro cartilage-like matrix production of embedded MSCs. PBMCs did produce significantly higher TNF-α and CCL2 in response to the GelMA with the highest endotoxin level compared to the other GelMAs. Although limited effects on chondrogenic differentiation were found in this study, caution with the use of commercial hydrogels is warranted in the translation from in vitro to in vivo studies because of regulatory constraints and potential inflammatory effects of the content of these hydrogels.

Tunable Cross-Linking and Adhesion of Gelatin Hydrogels via Bioorthogonal Click Chemistry

“…Gelatin represents an optimal choice to prepare ECM biomimetic hydrogels. In fact, being a collagen derivative, gelatin possesses unique advantages, compared to other synthetic and natural-derived polymers, including intrinsic presence of cell-adhesive motifs and MMP-sensitive sequences that guarantee cell adhesion and matrix remodeling, fundamental for cell-biomaterial interactions and potential tissue formation/regeneration. , Collagen source (e.g., porcine, bovine, fish) and its processing to extract gelatin (e.g., acidic or alkaline for type A and type B gelatin, respectively) can heavily influence the properties of the obtained gelatin hydrogels. , Thus, we selected type B gelatin as, compared to other gelatin types, it induces minor in vivo inflammatory response, and it exhibits lower immunogenicity and higher cytocompatibility. , Moreover, we prepared hydrogels using low-endotoxin gelatin, as controlled endotoxin levels must be guaranteed for potential translation from in vitro studies to in vivo applications, given regulatory constraints and endotoxin-related potential inflammatory effects …”

“…51,52 Moreover, we prepared hydrogels using low-endotoxin gelatin, as controlled endotoxin levels must be guaranteed for potential translation from in vitro studies to in vivo applications, given regulatory constraints and endotoxin-related potential inflammatory effects. 53 We selected a bioorthogonal cross-linking strategy based on the inverse-electron demand Diels−Alder reaction between Tz and Nb to cross-link the gelatin hydrogels we prepared. This cross-linking reaction is highly selective, it can occur in mild, cell-friendly conditions, 32−34 and it does not require that any external energy input and hydrogels can be easily prepared by simply mixing gelatin derivatives functionalized either by Tz or Nb.…”

Biofabrication of a shape-stable auricular structure for the reconstruction of ear deformities

“…The hydrogel gelMA supports chondrogenic differentiation of cartilage progenitor cells [ 36 , 56 ], is suitable as a bioink [ 57 ] and is available in clinical-grade quality [ 57 ]. Although the gelatin type A used in this hydrogel harbors endogenous endotoxins that may elicit an inflammatory response in more complex in vitro or in vivo environments [ 57 ], a recent study determined no critical effects of endotoxin level on chondrogenic differentiation [ 88 ]. The physicochemical properties of PCL enable additive manufacturing of porous 3D scaffolds that support construct integrity as well as neotissue formation [ 49 , 58 , 59 ].…”

The Influence of Bloom Index, Endotoxin Levels and Polyethylene Glycol Succinimidyl Glutarate Crosslinking on the Physicochemical and Biological Properties of Gelatin Biomaterials

“…It has been well-established in the literature that endotoxins are associated with macrophage activation [60,61] and macrophage aggregation is indicative of foreign body response [62][63][64]. Further, high endotoxin levels in gelatin preparations have been shown to significantly increase synthesis of TNF-a and CCL2 [65], notorious pro-inflammatory cytokines [66][67][68].…”