Characterization of a decellularized pericardium extracellular matrix hydrogel for regenerative medicine: insights on animal-to-animal variability

Abstract

In the past years, the use of hydrogels derived from decellularized extracellular matrix (dECM) for regenerative medicine purposes has significantly increased. The intrinsic bioactive and immunomodulatory properties indicate these materials as promising candidates for therapeutical applications. However, to date, limitations such as animal-to-animal variability still hinder the clinical translation. Moreover, the choice of tissue source, decellularization and solubilization protocols leads to differences in dECM-derived hydrogels. In this context, detailed characterization of chemical, physical and biological properties of the hydrogels should be performed, with attention to how these properties can be affected by animal-to-animal variability. Herein, we report a detailed characterization of a hydrogel derived from the decellularized extracellular matrix of bovine pericardium (dBP). Protein content, rheological properties, injectability, surface microstructure, in vitro stability and cytocompatibility were evaluated, with particular attention to animal-to-animal variability. The gelation process showed to be thermoresponsive and the obtained dBP hydrogels are injectable, porous, stable up to 2 weeks in aqueous media, rapidly degrading in enzymatic environment and cytocompatible, able to maintain cell viability in human mesenchymal stromal cells. Results from proteomic analysis proved that dBP hydrogels are highly rich in composition, preserving bioactive proteoglycans and glycoproteins in addition to structural proteins such as collagen. With respect to the chemical composition, animal-to-animal variability was shown, but the biological properties were not affected, which remained consistent in different batches. Taken together these results show that dBP hydrogels are excellent candidates for regenerative medicine applications.

Keywords: animal-to-animal variability; bovine pericardium; characterization; decellularized extracellular matrix; hydrogel; regenerative medicine.

FIGURE 1

Heatmap of matrisome peptides found in three different batches of dBP hydrogels.

FIGURE 2

Rheological properties of dBP hydrogels. (A) Gelation kinetics. Time-sweep curves of batches dBP.1, dBP.2, and dBP.3 at different hydrogels concentrations. Data are presented as average of experimental triplicate mean. (B) Frequency sweep curves of batches dBP.1, dBP.2, and dBP.3 at different hydrogels concentrations. Data are presented as average of experimental triplicate mean and SD. (C) Viscosity of three different batches of 9 mg/mL dBP hydrogels. (D) Tube inversion of 9 mg/mL dBP gelation before (pre-gel) and after gelation (hydrogel), with macroscopic image of dBP hydrogel.

FIGURE 3

Injectability evaluation of 9 mg/mL dBP hydrogel. (A) Gelation kinetics of non-extruded (Non-ext.) and (Ext.) extruded 9 mg/mL dBP hydrogels derived from dBP.1, dBP.2, and dBP.3 are shown singularly. (B) Gelation kinetics of non-extruded and extruded dBP hydrogels presented as average data from three different dBP batches, with SD.

FIGURE 4

Morphological analysis of 9 mg/mL dBP hydrogels with SEM. (A) SEM images of different dBP hydrogel batches at 9 mg/mL. × 100 magnification, scale bar: 500 μm and × 1,000 magnification, scale bar 50 μm are shown. (B) Frequency distribution of 9 mg/mL dBP hydrogel pore size.

FIGURE 5

Percentage wet weight variation over time of 9 mg/mL different dBP hydrogels batches (dBP.1, dBP.2, and dBP.3) placed in enzymatic medium (Enz.) or PBS.

FIGURE 6

dBP cytocompatibility. MTS assay performed on hMSCs cultured on three different batches of 9 mg/mL dBP hydrogels at 1 and 7 days. *: Day 1 vs. Day 7, °: dBP vs. CTR, p < 0.05.