Abstract
BACKGROUND: Tendon tissue engineering requires biomimetic scaffolds and mechanical cues to direct mesenchymal stem cell differentiation toward tenogenic lineages. Bone marrow-derived mesenchymal stem cells (BMSCs), aligned nanofiber scaffolds, and cyclic uniaxial stretching can be used to create a functional engineered ligament tissue. AIM: To investigate the effects of aligned nanofiber scaffolds and cyclic stretch on BMSC tenogenesis for ligament engineering. METHODS: BMSCs were cultured on aligned and random poly-lactic acid nanofiber scaffolds under static and cyclic tensile conditions (0.5 Hz, 2% strain, 2 hours/day) for 7 days using a mechanical loading system (CFILLOAD-300). The Ras homolog gene family (Rho)-associated coiled coil-containing kinase (ROCK) inhibitor Y27632 was applied to explore its role in tenogenic differentiation. Scaffold morphology was assessed by scanning electron microscopy, while cell morphology, viability, and alignment were evaluated via confocal microscopy with F-actin and 4',6-diamidino-2-phenylindole staining. Tenogenic gene expression (collagen type I alpha 2, collagen type III alpha 1, tenascin C, and tenomodulin) was quantified by quantitative polymerase chain reaction, and ligament-related protein levels (collagen I, collagen III, tenascin C, and tenomodulin) were analyzed by western blot. RESULTS: Scanning electron microscopy revealed that aligned scaffolds provided consistent directional structure, whereas random scaffolds displayed a disordered fiber arrangement. Confocal microscopy showed that under static conditions, BMSCs on aligned scaffolds grew parallel to fiber alignment, while those on random scaffolds grew randomly. Under cyclic tensile strain, BMSCs on both scaffold types exhibited elongation along the direction of strain, adopting a spindle-shaped morphology. Cyclic uniaxial strain enhanced cell viability and metabolic activity based on CCK-8 assay results and upregulated ligament-specific gene and protein expression on aligned scaffolds compared to static conditions. BMSCs on aligned scaffolds under tensile strain showed the highest expression of tenogenic markers, suggesting a synergistic effect of scaffold alignment and mechanical loading. ROCK inhibition with Y27632 upregulated alternative signaling pathways (focal adhesion kinase and runt-related transcription factor 2), further promoting tenogenic differentiation. CONCLUSION: Aligned nanofiber scaffolds combined with cyclic tensile strain provide an optimal environment for guiding BMSC differentiation toward ligamentous lineages, as assessed by increased expression of ligament-specific markers. Mechanical stimulation (uniaxial stretching) significantly influences BMSC tenogenic differentiation, and the combined use of aligned nanofibers and tensile strain further enhances this effect. The ROCK pathway plays a regulatory role in this process, though its precise mechanisms require further investigation.