gms | German Medical Science

Objectives: The iliotibial band (ITB) contains longitudinal and crossing type I collagen fiber bundles with few specialized fibroblasts arranged in longitudinal rows. This robust tissue is suitable for anterior cruciate ligament (ACL) reconstruction. Hence, ITB-derived fibroblasts attract interest for ligament tissue engineering purposes. Fibroblasts were often harvested by explant cultures, however, ITB fibroblasts have so far barely been characterized. An understanding of cell emigration from cultured explants might allow to seed biomaterials with ITB fibroblasts.

ITB fibroblasts were characterized before, during, and after emigration from the ITB to get a deeper understanding of this process and to utilize their migratory capacity for scaffold seeding.

Methods: Human ITB tissue (derived from n=6 females, n=7 males, mean age: 42.2 y; obtained from forensic autopsy) and ACL tissue (derived from n=9 females, mean age: 50.1 y; derived from knee replacement surgery) were characterized histopathologically (using the scoring system of Hasegawa et al., 2013). Explant cultures were prepared from ITB tissue and maintained for up to 10 weeks. Cell survival in explants, monolayers and spheroids was visualized using live/dead staining. The protein expression profile of targets, typically expressed by ligamentocytes was tested such as types I-III collagen, elastin, lubricin, decorin, aggrecan, fibronectin, tenascin C, CD44, beta1-integrins, scleraxis, vimentin, F-actin and VEGF-A and was then compared with ACL fibroblasts. Since the migratory potential of intrinsic and extrinsic fibroblasts is highly important in tendon and ligament healing, alpha-smooth muscle actin (alphaSMA), a marker of migrative myofibroblasts, was visualized in situ in ITB and ACL tissue and in vitro in isolated cells. ITB fibroblasts were self-assembled into spheroids, which were subsequently used for colonization of polyglycolic acid (PGA) scaffolds.

Results and conclusion: In contrast to the ACLs, ITB tissue revealed only minor features of degeneration but shared with ACLs cell subpopulations expressing alphaSMA (3.2-59.8%). However, in both tissues, no significant correlation between degeneration and alphaSMA expression was detected. ITB fibroblasts survived for several months in explant culture, strongly expressed alphaSMA and build F-actin stress fibers during emigration, suggesting myofibroblast transition and continuously formed novel monolayers. VEGF-A could also be detected in emigrating ITB fibroblasts. They shared their expression profile for the tested matrix components (except for elastin), receptors, scleraxis and vimentin with ACL fibroblasts. They formed compact spheroids (25,000 cells/spheroid, diameter: 20-40 µm).

This study provides evidence that using self-assembled spheroids, the migratory capacity of myofibroblastic ITB cells can be utilized for colonizing biomaterials for ACL tissue engineering and to support ligament healing.