Porcine Decellularized Diaphragm Hydrogel: A New Option for Skeletal Muscle Malformations

Abstract

Hydrogels are biomaterials that, thanks to their unique hydrophilic and biomimetic characteristics, are used to support cell growth and attachment and promote tissue regeneration. The use of decellularized extracellular matrix (dECM) from different tissues or organs significantly demonstrated to be far superior to other types of hydrogel since it recapitulates the native tissue’s ECM composition and bioactivity. Different muscle injuries and malformations require the application of patches or fillers to replenish the defect and boost tissue regeneration. Herein, we develop, produce, and characterize a porcine diaphragmatic dECM-derived hydrogel for diaphragmatic applications. We obtain a tissue-specific biomaterial able to mimic the complex structure of skeletal muscle ECM; we characterize hydrogel properties in terms of biomechanical properties, biocompatibility, and adaptability for in vivo applications. Lastly, we demonstrate that dECM-derived hydrogel obtained from porcine diaphragms can represent a useful biological product for diaphragmatic muscle defect repair when used as relevant acellular stand-alone patch.

Biomaterials and Their Biomedical Applications: From Replacement to Regeneration

“…A relevant area of application of 3D printed objects is tissue engineering since scaffolds can be developed with both micro and macro porosity where cells are able to attach, grow and differentiate. In this context, bioprinting is an innovative technique in which live cells are printed along with other biomaterials, generally hydrogels [79][80][81][82]. The aim of bioprinting is the production of fully functional human organs and tissues on a large scale.…”

Decellularized Skeletal Muscles Support the Generation of In Vitro Neuromuscular Tissue Models

“…Cell myogenic identity was confirmed in standard culture conditions by the presence of MyoD-positive cells and by the presence of multinucleated myotubes in both proliferating and differentiating culture conditions (Figure 1A). According to the evidence that fibroblasts are necessary to drive dSkM repopulation by myogenic cell in vitro [16], human myogenic cells were combined with murine fibroblasts in a defined ratio [16], seeded within the dSkM, and co-cultured for 21 days (Figure 1B). Fluorescence stereomicroscope imaging of calcein incorporation demonstrated the viability of the cells (Figure 1B, left panels).…”

“…Based on our previous results and on the neurotrophic properties of dSkM [16,17], we generated the neuromuscular construct by co-seeding human myogenic cells and murine fibroblasts into the dSkM together with oSpC onto the scaffold (Figure 2A). Calcein liveimaging performed at the fluorescence stereomicroscope, confirmed the presence of viable cells after 21 days of co-culture (Figure 2B).…”

“…Despite the fact that monitoring Fluo-4 within the 3D neuromuscular construct was not possible due to thickness and autofluorescence of the construct, myotubes located to the margins between the spinal cord and the dSkM clearly showed physiological activity (Figure 2E and Supplementary Video S1) with quantifiable calcium spikes (Figure 2E, left panel). Based on our previous results and on the neurotrophic properties of dSkM [16,17], we generated the neuromuscular construct by co-seeding human myogenic cells and murine fibroblasts into the dSkM together with oSpC onto the scaffold (Figure 2A). Calcein live-imaging performed at the fluorescence stereomicroscope, confirmed the presence of viable cells after 21 days of co-culture (Figure 2B).…”

“…Moreover, dSkM tissue has unique trophic and topographic properties which enhance repopulation, alignment and differentiation of myogenic cells [1]. These advantages pave the way to various applications of dSkM in vitro, e.g., investigation of cell viability and infiltration upon long-term culture [12,13], evaluation of tissue ECM and drug interaction mimicking intramuscular injections [14], or the use of dSkM-based hydrogel for in vitro and in vivo studies [15,16]. Our recent work has demonstrated that the dSkM retains a multitude of structural and other proteins that can enhance the repopulation of seeded myogenic cells [17].…”