Material Characterization and Temporospatial Immune Response to Cryogenically Sintered Extracellular Matrix Particle Scaffolds in Volumetric Muscle Loss Injury

Introduction: Volumetric muscle loss (VML) from severe traumatic injury leads to extensive skeletal muscle damage that exceeds natural regenerative capacity and results in permanent loss of function. Traditional treatment with autologous tissue transfer can partially restore function but at the cost of donor site morbidity in addition to risk of graft failure. There is a significant need to develop off-the-shelf biomimetic materials that can direct skeletal muscle regeneration and restore function in VML injuries. Extracellular matrix (ECM) is a dynamic network of connective tissue that provides cell support, mediates the host response to injury, and maintains a native tissue environment. Biologic scaffold materials derived from decellularized ECM are used to promote wound healing by initiating a Type 2 inflammatory response, which has been recently leveraged for tissue regeneration applications. However, current injectable ECM scaffolds insufficiently fill 3D tissue defects and lack the porosity optimal for cell migration. By controlling the porosity and integrity of a decellularized ECM scaffold, we aim to optimize its architecture for 3D muscular regeneration and promote immune cell infiltration, tissue formation, and vascularization. We characterized cryogenically sintered ECM particle scaffolds and investigated their site-appropriate immune response in VML injuries.

Materials and

Methods: Porcine-derived small intestinal submucosa (SIS) was mechanically scraped and decellularized using peracetic acid and ethanol. The resulting SIS was lyophilized, verified decellularized (dsDNA < 200 ng/mg), and milled into particles. This particulate SIS was hydrated at high concentrations within a cyromold, frozen, and lyophilized to create a porous 3D scaffold where individual particles are sintered via physical interactions, which we term “cryogenic particle sintering”. We examined 50, 100, and 200 mg/mL to determine the effect of concentration, and freezing rate starting from room temperature to -30°C to -196°C to control pore size. Scaffold porosity was assessed using quantitative ImageJ analysis of scanning electron microscopy (SEM) images, and relative compressive strength was measured using an RSA-G2 Solids Analyzer. Scaffold stability was visualized with a disintegration assay, where scaffolds were submerged in PBS on an orbital shaker at 37°C for 16 days. In vitro scaffold cytocompatibility was determined by seeding mouse 3T3 fibroblasts onto the scaffolds; cell viability was quantified using a CellTiter-Glo 3D Cell Viability Assay and calcein-AM/ethidium homodimer staining with confocal microscopy. In vivo remodeling and immune response were studied following implantation of SIS-ECM scaffolds or particles after a VML injury in 8-week-old C57BL/6J mice. Approximately 3x4x4 mm of quadricep muscle was removed and replaced with SIS-ECM. Temporospatial characterization of immune and remodeling response to the scaffold was assessed by histology while quadriceps and inguinal lymph nodes were harvested for qRT-PCR gene transcription. Genes of interest included IFN-γ for a Th1 immune response, IL-4 for Th2, and IL-17A for Th17.

Results, Conclusions, and Discussions: SEM confirmed that ECM particle concentration and freezing rate controlled scaffold porosity. % porosity decreased with increasing concentration, from 69% for 50 mg/mL to 61.5% for 200 mg/mL. Fabrication at -80°C resulted in the highest % porosity of 67.3% compared to other freezing rates. Mechanical testing showed a similar dependency on concentration and freezing rate; compressive strength increased 1.5-fold from 50 mg/mL to 200 mg/mL, and 5-fold from -196°C to -30°C. The SIS-ECM scaffold maintained its macrostructure following a 16-day disintegration experiment. SIS-ECM scaffolds successfully recruited 3T3 cells and encouraged cellular infiltration and proliferation, as evidenced by the density of spindle-shaped cells. 3T3 cell viability was enhanced in 50 and 100 mg/mL scaffolds during a 1, 3, and 7-day period compared to 200 mg/mL scaffolds, such as a 9.6-fold change in cell viability for 50 mg/mL and 9.3-fold change for 100 mg/mL compared to 200 mg/mL at day 7. Confocal microscopy showed that the 50 and 100 mg/mL scaffolds had plentiful fibroblast adhesion. % live cells for the 50 mg/mL scaffold increased from 62.4% on day 1 to 82.5% on day 7 and for 100 mg/mL rose from 67.2% to 82.3%, while the 200 mg/mL showed a lower viability, increasing from 48.5% to 68.2%. In a VML injury model in vivo, there was substantial immune cell infiltration into the SIS-ECM scaffold implant and the nearby musculoskeletal tissue by day 7 and day 14. Particles were unevenly distributed and compressed within the tissue deficit site while scaffolds maintained a porous structure and defined scaffold-tissue interface. Scaffolds induced a 139-fold increase in quadricep IL-4 expression compared to saline alone at day 14, confirming that fundamental SIS-ECM bioactivity was retained after scaffold fabrication.
In conclusion, these findings show that we can manipulate the physical and structural properties of cryogenically sintered ECM particle scaffolds by changing concentration and temperature to subsequently alter mechanical and biological activity. These engineered scaffolds are mesoporous and retain immunomodulatory bioactivity in vivo. This study shows that ECM material can be processed into complex 3D structures that may be suitable as readily available scaffolds for muscular tissue engineering.

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