MSC Origin and Biomechanical Conditioning Determine ECM Maturation in Tissue-Engineered Matrix

Background: The extracellular matrix (ECM) plays a central role in the mechanical strength and functional integration of tissue-engineered matrix (TEM), particularly in cardiovascular and load-bearing applications. Mesenchymal stromal cells (MSCs) from different sources may vary in their ECM-forming potential. Methods: In this study, adipose-derived (hADMSC), bone marrow-derived (hBMSC), and umbilical cord-derived MSCs (hUCMSC) were compared with human dermal fibroblasts (HDFBs) as a reference. Cells were seeded onto polyglycolic acid (PGA)/poly-4-hydroxybutyrate (P4HB) scaffolds and cultured for 3 weeks under static or hydrodynamic conditions using orbital shaking. TEM development was assessed macroscopically, histologically (using H&E and Masson's trichrome stains), and by polarized light microscopy (Picrosirius Red), alongside biochemical assays that quantified DNA, glycosaminoglycan (GAGs), and hydroxyproline (HYP). Results: Hydrodynamically stimulated culture consistently improved ECM deposition across all groups. TEMs exposed to hydrodynamic stimulation (hydrodynamic conditions) were thicker, more uniformly filled, and exhibited increased collagen deposition compared with static TEMs, which remained thinner and showed persistent scaffold remnants. Polarized light analysis demonstrated that dynamic loading promoted collagen maturation in all groups, as evidenced by an increased prevalence of thick, birefringent collagen fibers indicative of mature collagen. Biochemical analyses showed that HDFB-derived TEMs produced the highest total collagen and ECM content under both static and hydrodynamic conditions; however, these matrices remained comparatively thin and densely packed. In contrast, MSC-derived TEMs formed thicker and more spatially distributed ECM in response to hydrodynamic stimulation. Conclusion: Among the MSC sources, hUCDMSC-derived TEMs exhibited the most advanced collagen maturation and the most uniform collagen distribution under hydrodynamically stimulated culture, whereas hADMSC-derived TEMs showed the greatest matrix thickening and volumetric ECM expansion with intermediate collagen maturation. hBMSC-derived TEMs displayed clear responsiveness to hydrodynamic stimulation but remained limited in overall collagen deposition and fiber maturation. These findings underscore that both hydrodynamic stimulation and cell source are critical not only for maximizing ECM deposition, but also for ensuring physiologically relevant collagen maturation and matrix organization in grafts suitable for clinical translation.