Abstract
Background: Human mesenchymal stem cells (hMSCs) possess potential of bone formation and were proposed as ideal material for tissue regeneration against osteoporosis. Although a plethora of elucidation regarding the directing effect on lineage specification by physical cues has been proposed, how mechanical stimulations impact the intracellular viscoelasticity during osteo-lineage commitment remained enigmatic. Methods: Cyto-friendly 3D matrix was prepared on soft-lithographed device. The substrate was basically polyacrylamide and conjugated with fibronectin. Stiffness of the scaffolds was determined by the mixing ratio of monomer and crosslinker. The hMSCs were injected with fluorescent beads by gene gun before being cultured in the matrix and chemically induced toward osteogenesis. The mechanical properties were assessed using video particle tracking microrheology. On post-induction day 0, 7, 14, 21, inverted epifluorescence microscope with charge-coupled camera was exploited to capture the projected Brownian trajectory of indwelling hMSCs. Mean square displacement thereof was calculated and transformed into intracellular viscoelasticity by generalized Stokes-Einstein equation.Results: Two different stiffness of 3D culture microspheres (12 kPa, 1 kPa) were established. A total of 45 cells were assessed. Initially, hMSCs possessed equivalent mechanical traits in the first week. Nonetheless, cells cultured in rigid matrix displayed statistically significant elevation over elastic (G’) and viscous moduli (G”) on day 7 (G’: 192±8 vs. 114±8, p<0.01; G” 204±9 vs. 169±10 Pa, p<0.01) and day 14 (G’: 190±6 vs. 129±7, p<0.01; G” 222±10 vs. 161±9 Pa, p<0.01). To appreciate the trend of fluctuation, subsequent measurements were compared with the respective modulus at day 0. The soft niches no longer facilitated stiffening hMSCs, whereas the effect by rigid substrates was consistently during the entire differentiation course.Conclusions: Stiffness of the matrix impacted the viscoelasticity of hMSCs. Detailed elucidation on how microenvironment impacts mechanical properties and differentiation of hMSCs will facilitate the advancement of tissue engineering and regenerative medicine.
Alteration of 3D Matrix Stiffness Regulates Viscoelasticity of Human Mesenchymal Stem Cells