Strain Distribution Across the Myotendinous Junction and its Role in the Regeneration of the Junction

Abstract

The muscle tendon junction (MTJ) is a specialised interface between the muscle and the tendon, which transmits the force from the muscle to the tendon. Injuries at the MTJ are common with 28% of injuries in the muscle-tendon-bone unit occurring at the MTJ. These strain and tear injuries limit the ability of patients to move, affecting their quality of life. For a complete tear of the MTJ, surgical intervention is the only treatment; however, satisfactory long-term outcomes after surgery are limited and tears often reoccur. Therefore, investigating the mechanical behaviour and regeneration of the MTJ will shed light on how healing, repair and regeneration can be promoted. In order to understand the mechanical behaviour of the MTJ, the full-field strain distribution of the MTJ was reported. Using digital volume correlation (DVC) combined with X-ray computed tomography (XCT), the MTJ was imaged and the changes in strain under a tensile load (2.4 N) were measured. High strain (reaching 12, 16, and 12% for the 1st principal stain (εp1), shear strain (γ), and von Mises strain (εVM) respectively) was measured at the MTJ and these values reduced into the body of the muscle and into the tendon, which indicated that the MTJ was at risk of being damaged in activities associated with high strain such as excessive physical activities. However, the XCT imaging process caused dehydration of the specimens, and the effect of shorter scan time was investigated using confocal microscopy. Images were taken under preload (0.4 N), 0.8 N, and 1.2 N load, and high strain concentration was observed at the junction at both 0.8 N and 1.2 N load for εp1, γ and εVM, and the strain values reached 15, 34 and 19% respectively.
In order to understand the effects of strain on the regeneration of the MTJ, aligned electrospun polycaprolactone fibres were fabricated, and human myoblasts and tenocytes were cultured on the fibres. The application of 10% cyclic strain significantly increased cell elongation, and the gene and protein expression of paxillin and collagen 22, naturally found in the MTJ and known to be MTJ markers, was induced. Co-culture of myoblasts and tenocytes on scaffolds subjected to strain induced higher MTJ marker protein expression compared with myoblasts and tenocytes cultured separately on scaffolds subjected to strain. For the first time, these results showed that the combination of the strain and co-culture of myoblasts and tenocytes can promote MTJ marker gene expression and production of proteins. However, in these in vitro constructs, no differentiation cue was added, and there was no spatial organisation of the cells into a typical structure of an MTJ. Therefore, decellularised extracellular matrix (DECM) derived from sheep MTJs was used as a scaffold in order to provide the native structure of the MTJ with potentially the appropriate mechanical properties and differentiation cues. Human mesenchymal stem cells (MSCs) were seeded on DECM and 10% cyclic strain was applied using a bioreactor. MSCs with DECM showed significantly higher gene and protein expression of MTJ markers than MSCs in 2D culture. Although collagen 22 protein expression was higher for cells cultured on DECM subjected to strain than for those without strain, reduced MTJ marker gene expression (collagen 22 and paxillin) was observed when the strain was applied. In addition, tissue specific differentiation was observed in MSCs, which showed myogenic differentiation on DECM from the muscle, and tenogenic differentiation on DECM from the tendon. For the first time, these results showed that DECM derived from MTJs can induce MTJ marker gene and protein expression from MSCs, however, the effect of strain on the MTJ development in DECM culture needs further investigation.
Overall, the data presented in this thesis suggested that the strain concentration was high at the MTJ under a uniaxial tensile load, which showed that the MTJ was at higher risk of being damaged in activities associated with excessive physical activity. In addition, the data also showed that co-culture of myoblasts and tenocytes, strain, and differentiation factors were all essential to develop the MTJ, however, further investigation is needed in order to develop more mature MTJ in vitro.

Date of Award	24 Jun 2024
Original language	English
Awarding Institution	University of Portsmouth
Supervisor	Gordon Blunn (Supervisor), Marta Roldo (Supervisor) & Aikaterina Karali (Supervisor)