Elucidating the Mechanism of Gelation for Decellularized Extracellular Matrix Hydrogels

Naturally derived, decellularized extracellular matrix-based hydrogels (ECM hydrogels) have been increasingly investigated as biomaterial scaffolds for tissue repair and regeneration in a variety of disease models due to their injectability through minimally invasive methods, tunable properties, ability to assemble and form a hydrogel <i>in situ</i>, and intrinsic regenerative properties. Most commonly, their rheological, mechanical, optical properties, and protein composition have been studied to understand the gel’s stability, gel formation kinetics and the extracellular matrix protein component type and distribution. Natively within mammals, type I collagen is the most abundant structural fibrillar protein and forms a hydrogel through fibril self-assembly and crosslinking when it is pure and has been assumed to be the main driver for hydrogel formation within heterogeneous ECM hydrogels. The goal of this study was to provide more fundamental materials characterization of ECM hydrogels and determine whether type I collagen is in fact the driver for ECM hydrogel formation.<br/><br/>Using three batches of a porcine derived decellularized myocardial ECM hydrogel, we first ensured batch consistency by quantifying residual dsDNA, quantifying sGAG concentration and rheological measurements. We also employ Fourier transform infrared spectroscopy (FTIR) to analyze the bonds within our ECM hydrogels, thermal gravimetric analysis (TGA) from 30 to 700°C to analyze degradation temperatures, and turbidimetry analysis to observe gelation kinetics. Finally, using cryogenic transmission electron microscopy and two photon microscopy, we analyze the fibril structures within our ECM hydrogel and quantify the differences in fibril diameters before and after incubation at 37°C.<br/><br/>All batches contained less than 1 ng DNA per mg of ECM showing adequate decellularization and 10 µg of sGAGs per mg of ECM. We observed consistent complex viscosity across all samples that had not been incubated, and similar storage and loss moduli values for gels after 24 hours of incubation. The FTIR revealed the same peptide-related peaks (amides I, II and III) at similar peak intensity ratios also seen in a pure type I collagen control. TGA of our ECM hydrogel revealed degradation onset temperatures around 304.6±1.7°C and 624.9±16.8°[KC1] C, consistent with literature reported values for collagen. Turbidimetry analysis revealed similar kinetics of gelation evidenced through similar times to achieve 50% of maximum equilibrium absorbance at around 15 minutes. ECM hydrogel batches were imaged before and after incubation at 37°C for 24 hours using two photon microscopy through second harmonics generation and two photon fluorescence, which leveraged the intrinsic property that only fibrillar, helical collagens produce signal. Incubated samples revealed distributed linear fibers in a grid-like conformation signifying the formation of a hydrogel network. Using cryogenic transmission electron microscopy, we analyzed fiber diameters within our samples for fresh, unincubated samples and samples incubated at 37°C for 24 hours. Fibers in fresh samples averaged 45.6 ± 21.7 nm, 48.7 ± 10.8 nm, and 43.7±15.5 nm in diameter across batches showing consistency. Incubated samples averaged significantly higher fiber diameters when compared to their fresh counterparts at 113.1±48.2 nm, 156.1±79.3 nm and 128.9±56.8 nm, respectively, where all measured fibers within the incubated group revealed a distinct 67 nm D-periodicity unique to fibrillar collagens. Combined, these data reveal batch to batch consistency using more sensitive materials characterization techniques for the ECM hydrogel while also providing visual evidence, through two microscopy techniques, that type I collagen self-assembly occurs during incubation at 37°C and is one of the main drivers of ECM hydrogel formation.