Validation of FC on in silico vector fields performed as expected under controlled conditions. Perfectly aligned vectors produced minimal FC values while increasing levels of disruption were reflected by increasingly higher FC values. FC was also able to capture isolated angular disruptions and identify transition points where FC values changed abruptly. This confirmed the metric’s sensitivity to local deviations from uniform collagen alignment.

Following application of FC to MADI-derived vector fields from the human ACL samples, distinct differences between healthy and injured ACL tissue were observed (Figure 4). A centrally located band of consistently low FC values extended longitudinally through the ligament measuring 34x6x4mm. The presence of this band aligns with previous literature detailing dimensions of a flat, central mid-substance with straight, uniform collagen alignment native to healthy ACLs [16,17]. This organised central band was absent in partially torn and completely torn ACL samples. These samples displayed a more heterogenous and spatially disordered FC distribution throughout the tissue volume. The final reconstructed ACL sample exhibited partial re-emergence of the central band which may suggest some restoration of native collagen structure following surgical repair[18].

Quantification of FC values supported these qualitative observations. Healthy and reconstructed ACLs demonstrated greater global spatial autocorrelation of FC values (Moran’s I = 0.25 and 0.21 respectively), compared to partially torn and completely torn ACL samples (Moran’s I = 0.08 and 0.17 respectively).

FC colourmaps were successfully superimposed onto streamline tractography (Figure 5). Composite images displaying fibre pathway and local organisational characteristics were thereby created. These integrated visualisations maintained the patterns highlighted in our 3D scatter plots. Healthy ACL contained its central coherence which persisted throughout the length of the ACL. However, pathological displays kept their heterogenous patterns.