Hypoxia-Induced Extracellular Matrix Deposition in Human Mesenchymal Stem Cells: Insights from Atomic Force, Scanning Electron, and Confocal Laser Microscopy

(1) Background: The extracellular matrix (ECM) is a natural scaffold for cells, creating a three-dimensional architecture composed of fibrous proteins (mainly collagen) and proteoglycans, which are synthesized by resident cells. In this study, a physiological hypoxic environment was utilized to enhance ECM production by human mesenchymal stem cells (hMSCs), a process relevant to tissue engineering and regenerative medicine. (2) Methods: hMSCs were treated with deferoxamine (DFO), a pharmaceutical hypoxia-mimetic agent that induces cellular responses similar to low-oxygen conditions through stabilization of hypoxia inducible factor-1α (HIF-1α). The time points 0 h 24 h, 3 h 24 h, and 24 h 24 h refer to DFO being added immediately after cell seeding (before cells adhesion), 3 h after cell seeding (during initial cells attachment), and 24 h after cell seeding (after focal adhesions formation and actin organization), respectively, to evaluate the influence of cell adhesion on ECM deposition. hMSCs incubated in culture media were subsequently exposed to DFO for 24 h. Samples were then subjected to cell viability tests, scanning electron microscopy (SEM), atomic force microscopy (AFM) and laser scanning confocal microscopy (CLSM) assessments. (3) Results: Viability tests indicated that DFO concentrations in the range of 0–300 µM were non-toxic over 24 h. The presence of collagen fibers in the DFO-derived ECM was confirmed with anti-collagen antibodies under CLSM. Increased ECM secretion was observed under the following conditions: 3 μM DFO (24 h 24 h), 100 μM DFO (0 h 24 h) and 300 μM DFO (3 h 24 h). SEM and AFM images revealed the morphology of various stages of collagen formation with both collagen fibrils and fibers identified. (4) Conclusions: Our preliminary study demonstrated enhanced ECM secretion by hMSC treated with DFO at concentrations of 3, 100, and 300 µM within a short cultivation period of 24–48 h without significant affecting cell viability. By mimicking physiological processes, it may be possible to stimulate endogenous tissue regeneration, for example, at an injury site.