Effects of extracellular matrix, cytokines, trophic factors, and cellular interactions on satellite cell activation in musculoskeletal diseases

Abstract

Skeletal muscle regeneration depends on satellite cells (SCs) integrating biochemical and biophysical signals from their microenvironment (Yin, Price and Rudnicki, 2013;Gilbert et al., 2010). This thesis defines how extracellular matrix (ECM) compositions including Laminin, Collagen Type-1 (CT-1) and Fibronectin, and synthetic substrates such as Poly-L Lysine (PLL) and the Laminin-mimetic substrate, PhenoDrive-Y (PDY),transient inflammatory priming and neighbouring cells regulate human SC adhesion, phenotypic stability, myogenic progression, and early angiomyogenic co-culture behaviour. Baseline characterisation of Innoprot-derived human SC confirmed marked heterogeneity at passage 1, with Pax7-enriched population (57.3%Pax7+) and a corresponding (42.7% Pax7- population), and only a negligible MyoD1+fraction (0.59% of total events), establishing a platform for testing how culture conditions determine quiescent and myogenic differentiation states.
Across ECM conditions, Laminin, CT-1, Fibronectin, PLL, and the Laminin-mimetic substrate (PD-Y), time-lapse and visible light microscopy, confocal immunostaining (Pax7, MyoD1, Itg-α7, Notch1, Desmin, Rhodamine-Phalloidin), flow cytometry (Pax7/MyoD1), and the trophic-factor output of Insulin growth factor-1 (IGF-1) were examined to determine substrate-dependent responses. Laminin and PD-Y consistently promoted earlier acquisition of elongated, aligned morphology and myotube/ myofibre-like organisation compared with CT-1, while supporting coexisting Pax7+ and MyoD1+ populations at Day 14 and 30. In parallel, IGF-1 secretion was detectable and its levels was significantly higher and time-dependent on PLL, PD-Y and laminin, but remained undetectable on CT-1 and Fibronectin, suggesting that trophic support is associated with enhanced myogenic structural organisation.
Prolonged culture showed that Laminin and PD-Y supported more stable integrinassociated and structural maturation, characterised by strong, widespread Itg-α7expression and increasingly organised Desmin and F-actin architecture by Day 30. By contrast, CT-1 and Fibronectin exhibited more heterogeneous organisation, with a gradual reduction in ordered actin and adhesion-associated features over time. Acute inflammatory priming for 48 hours with IL-1β or TWEAK, followed by removal at Day2, revealed a substrate-dependent difference in the maintenance of commitment to differentiation markers, with MyoD1 loss most pronounced on CT-1, partially observed on Laminin when introduced to IL-1β, and more consistently preserved on PD-Y. Notably, Desmin and F-actin maturation could persist even when MyoD1 was weak or absent at later time points. Moreover, the co-culture of SCs and HUVECs further demonstrated that ECM composition is a controllable parameter for early angiomyogenic organisation. Laminin supported reproducible niche-like organisation, PD-Y enhanced the definition of capillary-like networks, and CT-1 was the least supportive of coordinated organisation, consistent with the influence of endothelial microenvironmental signals on SC progression at early time points.
Collectively, these findings identify basement-membrane-like substrates, particularly PD-Y and laminin, as a rational basis for designing reproducible muscle-tissue engineering platforms that support SC expansion and differentiation, maintain resilience to inflammatory perturbation, and facilitate vascular integration.

Date of Award	Jul 2026
Original language	English
Awarding Institution	University of Brighton
Supervisor	Matteo Santin (Supervisor), Angela Sheerin (Supervisor) & Laila Kudsiova (Supervisor)