Uniaxial Cyclic Tensile Stretching at 8% Strain Exclusively Promotes Tenogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stromal Cells

Abstract

The present study was conducted to establish the amount of mechanical strain (uniaxial cyclic stretching) required to provide optimal tenogenic differentiation expression in human mesenchymal stromal cells (hMSCs) in vitro, in view of its potential application for tendon maintenance and regeneration. Methods. In the present study, hMSCs were subjected to 1 Hz uniaxial cyclic stretching for 6, 24, 48, and 72 hours; and were compared to unstretched cells. Changes in cell morphology were observed under light and atomic force microscopy. The tenogenic, osteogenic, adipogenic, and chondrogenic differentiation potential of hMSCs were evaluated using biochemical assays, extracellular matrix expressions, and selected mesenchyme gene expression markers; and were compared to primary tenocytes. Results. Cells subjected to loading displayed cytoskeletal coarsening, longer actin stress fiber, and higher cell stiffness as early as 6 hours. At 8% and 12% strains, an increase in collagen I, collagen III, fibronectin, and N-cadherin production was observed. Tenogenic gene expressions were highly expressed (p < 0.05) at 8% (highest) and 12%, both comparable to tenocytes. In contrast, the osteoblastic, chondrogenic, and adipogenic marker genes appeared to be downregulated. Conclusion. Our study suggests that mechanical loading at 8% strain and 1 Hz provides exclusive tenogenic differentiation; and produced comparable protein and gene expression to primary tenocytes.

Figure 1

Characterization of hMSCs was confirmed. (a) Density gradient separation of human bone marrow: (A) plasma, (B) mononuclear cells, (C) ficoll paque, (D) erythrocytes, and granulocytes. (b) Morphology of replated cells showed homogeneous and fibroblastic shape. (c) Representative images of multicolour CD markers by flow cytometry. The results showed that hMSCs expressed at least 90% of double-positive expression, double-negative, or coexpressed positive and negative markers. (d) Trilineage differentiation potential of the hMSCs into osteogenic, adipogenic, and chondrogenic lineage.

Figure 2

Microscopy images of unstrained and strained hMSCs. (a) Phase-contrast photomicrographs of hMSCs subjected to cyclic uniaxial stretching in different magnitude and duration of stretching. (b) Higher magnification of phase contrast of unstrained and 8% strained hMSCs at 72 h and tenocytes. (c) Confocal laser scanning micrographs showing actin stress fibers (green) and nuclei (blue) of unstrained cells and 4%, 8%, and 12% strained cells at 72 h. The substrate was stretched in the red arrow direction.

Figure 3

Biochemical analysis of MSCs subjected to various mechanical stimuli for different duration of stimulation. Content of (a) total collagen, (b) GAG, and (c) elastin of strained cells was measured to determine the total quantity of the respective ECM component in the sample which released to medium. (d) The level of collagen type I in the medium was measured by ELISA. The ratio of the ECM expression was counted by normalizing to the expression amount of corresponding unstrained groups (indicated as 1). Significance p < 0.05 was represented by ^∗ which compared to unstrained. N = 6, n = 3, error bar ± SD.

Figure 4

ECM expression on unstrained and strained cells. (a) Comparison of different collagen staining on various mechanical stimuli hMSCs at 72 h and tenocytes as positive control. (b) Immunofluorescence staining of fibronectin and N-cadherin on unstrained and strained hMSC for 6 h or 72 h. The expression of fibronectin and N-cadherin was enhanced by the cyclic stretch and magnitude strain dependent. The substrate was stretched in the red arrow direction. (c) Thicker fibronectin fibrils were formed by cyclic mechanical stimulation.

Figure 5

Mechanical stretching altered hMSC surface antigen expression but did not express osteogenesis and adipogenesis. (a) Expression levels of the CD markers of hMSCs cultured in mechanical stretching with different strains. Significance (p < 0.05) was represented by ^∗ which compared to unstrained. n = 3, error bar ± SD. (b) Percentage of multicolour expression for lymphocyte adhesion molecule CD44 and endoglin CD105. Fluorescent expression intensity and area of CD44 and CD105 in (A) 4%, (B) 8%, and (C) 12% strain magnitudes. (c) Representative images of Alizarin Red- and Oil Red O-stained hMSCs: (A) positive Alizarin Red staining on osteogenic medium cultured hMSCs, (B) negative Alizarin Red result on mechanical stimulated hMSCs, (C) positive Oil Red O staining on adipogenic medium cultured hMSCs, (D) negative Oil Red O result on strained hMSCs.

Figure 6

The comparison of cell surface topography between the unstrained hMSCs, strained hMSCs, and tenocytes, visualized by AFM. (a) Representative AFM height and deflection scans of unstrained hMSCs and 4%, 8%, and 12% strained hMSCs, and tenocytes. In height images, brighter colour indicates higher distance of substrate. In deflection images, the detailed structure of presumably the stress fiber could be observed with AFM in different cell groups. The direction of uniaxial strain was in the red arrow direction. (b) Young's modulus on the cytoskeleton of the cells subjected to 4%, 8%, or 12% cyclic stretching for 72 h as indicated. The ratio was counted by normalizing to the expression amount of corresponding unstrained groups (indicated as 1). Statistical significance (p < 0.05) was represented by ^∗ relative to the unstrained group. n = 3, error bar ± SD.

Figure 7

mRNA expression level of different genes subjected to different strain for different time point. (a) mRNA expression of MMP3 and PRR16. (b) ECM component (COL1, COL3, and DCN). (c) Tendon cell lineage (TNC, SCX, and TNMD). (d) Bone cell lineage (RUNX2, ALP, and OCN). (e) Cartilage cell lineage (COL2, SOX9, and COMP). (f) Adipose cell (PPARG) and smooth muscle cell (TAGLN). The expression level of each gene was normalized with the level of housekeeping gene. The value of fold change was presented as the ratio of strained group with unstrained group. Statistical significance (p < 0.05) was represented by ^∗ which compared to unstrained. N = 6, n = 3, error bar ± SD.