3.1 Increased cell spreading area promoted osteogenesis and inhibited adipogenesis of BMSCs through F-actin assembly
We modulated cell spreading area through different concentrations of fibronectin (FN) at 5 × 104/ml of MSCs on tissue culture plates (TCPs). FN is the adhesive protein exists ubiquitously in ECM that cells attach to integrin and actin cytoskeleton (Kilian et al, 2012), so we explored whether FN could regulate cell morphology and BMSCs differentiation on TCPs, which are stiffer more than several MPas.
For BMSCs differentiation, initially with 0 μg/ml of FN, cells exhibited both fat vacuoles and ALP staining, and as FN concentration increased to 5,10,20 μg/ml, more pronounced ALP staining and fewer fat vacuoles were exhibited, indicating BMSCs commit higher extent of osteogenesis and lower adipogenesis (Fig 1A). Also, Rt-PCR illustrated an increase of osteogenic markers (RUNX2 and OCN) and a decrease of adipogenic markers (PPAR-γ2 and CEBP-α) along with higher FN concentration (Fig 1B). These results indicated that larger spreading area directed BMSCs into higher extent of osteogenesis and lower adipogenesis. Meanwhile, immunofluorescence staining indicated that an increase of FN concentration significantly promoted filamentous actin assembly and cell spreading area (Fig 2).
Then, we investigated the underlying role of F-actin in BMSCs differentiation induced by FN. On TCPs with 0 µg/ml of FN at a seeding density of 5 × 104 /ml (group FN0), after 14 days of adipo-osteo co-induction, cells exhibited both fat vacuoles and ALP deposition (Fig 3). Cytochalasin D is an inhibitor of F-actin polymerization and elongation. In the group that BMSCs cultured with actin polymerization inhibitor cytochalasin D and 0 µg/ml of FN (group FN0+CD), we found cells exhibited mainly fat vacuoles and less ALP deposition (Fig 3A) and PPAR-γ2 and CEBP-α exhibited the highest levels in all groups, indicating osteogenesis of BMSCs was inhibited by CD. In the group that BMSCs cultured with 10 µg/ml of FN and CD (group FN10+CD), FN rescued osteogenesis of BMSCs and inhibited adipogenesis compared with group FN0+CD, as more ALP and fewer fat vacuoles were exhibited and RUNX2 and OCN mRNA levels were higher than group FN0+CD (Fig 3). These results illustrated that FN increased cell spreading area and promoted osteogenesis of BMSCs through promoting F-actin assembly.
3.2 Interplay of effects of FN and matrix stiffness on BMSCs differentiation
Cytosoft® plates with elasticity moduli of 0.5 kPa and 32 kPa were chosen for they respectively resembled rigidity of adipose tissue and pre-calcified bone and have been proven to induce BMSCs into adipogenesis and osteogenesis [10]. As we have verified the effects of FN in regulation of cell spreading area, we seeded BMSCs on soft and stiff substrates coated with different concentrations of FN to modulate cell spreading areas.
First, we found increase of FN concentrations on both 0.5 kPa and 32 kPa substrates induced higher osteogensis and lower adipogenesis of BMSCs (Fig 4). Cells exhibited more pronounced ALP deposition and fewer oil droplets as FN concentration increased from 100 to 300 µg/ml on 0.5 kPa substrates, and from 50 to 100 µg/ml on 32 kPa substrates (Fig 4A). Also, PCR results showed OCN and RUNX2 levels increased while CEBP-α and PPAR-γ levels decreased when FN concentrations increased on both substrates (Fig 4B). Meanwhile, immunostaining results showed cells manifested more spreading morphology as FN concentrations increased on both substrates (Fig 5A).
Second, we found when cell spreading area reached similar levels on 0.5 kPa and 32 kPa substrates through different concentrations of FN, cell differentiation of BMSCs didn’t manifest significant difference either. We coated 300 µg/ml of FN on 0.5 kPa substrates(FN300/0.5 kPa group) to achieve similar cell spreading areas with cells on 32 kPa substrates coated with 100µg/ml of FN(FN100/32 kPa group), and also we coated 30 µg/ml of FN on 32 kPa (FN30/32 kPa group) to reach similar cell spreading areas with cells on 0.5 kPa substrates coated with 100µg/ml of FN(FN100/0.5 kPa group), as shown in immunostaining of F-actin in Fig 5A. Then we found when cell spreading areas reached similar levels, cells showed similar extent of osteogenesis and adipogenesis on substrates of different stiffness (Fig 4). Cells in both FN300/0.5 kPa group and FN100/32 kPa group showed mainly ALP staining and few fat vacuoles, indicating BMSCs mainly commit osteogenesis (Fig 4A). PCR results showed consistent results, as OCN and RUNX2 mRNA showed similarly high levels in both groups without significant difference, and CEBP-α and PPAR-γ levels showed similarly low levels (Fig 4B). On the contrary, cells in both FN100/0.5 kPa group and FN30/32 kPa group showed little ALP deposition and many fat vacuoles (Fig 4A), and OCN and RUNX2 mRNA showed similarly low levels in both groups without significant difference, and CEBP-α and PPAR-γ levels showed similarly high levels (Fig 4B). These results indicated when cell morphology and matrix stiffness exerted antagonistic effects, the former took over. Also, when cell spreading area was set, increase of matrix stiffness failed to exert significant effects on osteogenesis and adipogenesis of BMSCs.
Finally, we found matrix stiffness could exert effects on osteogenesis and adipogenesis of BMSCs only when cell morphology simultaneously adapted. Coated with the same FN concentration of 100 µg/ml, matrix stiffening from 0.5 kPa to 32 kPa still prominently promoted osteogenesis and suppressed adipogenesis, as more ALP and fewer fat vacuoles were exhibited (Fig 4A). Also, PCR results indicated significantly higher OCN, RUNX2 and lower CEBP-α and PPAR-γ levels as matrix stiffening (Fig 4B). More importantly, cells simultaneously spread to larger areas when matrix stiffness increased, as indicated in immunostaining of F-actin (Fig 5A).
3.3 YAP/TAZ activity of BMSCs under regulation of FN and matrix stiffness
First, we found increase of FN concentration on both 0.5 kPa and 32 kPa substrates induced nuclear translocation of YAP/TAZ of BMSCs. As shown in Fig 5B, YAP/TAZ mainly localized in cytoplasm on 0.5 kPa substrates coated with 100 µg/ml of FN and on 32 kPa coated with 30 µg/ml of FN, while YAP/TAZ translocated to nuclei on 0.5 kPa substrates coated with 300 µg/ml of FN and on 32 kPa coated with 10 µg/ml of FN.
Second, when cell spreading area reached similar levels on 0.5 kPa and 32 kPa substrates through respectively 300 µg/ml and 30 µg/ml of FN, YAP/TAZ immunostaining also manifested similar intensity and localization in both nuclei and cytoplasm. As shown in Fig 5B, in both FN300/0.5 kPa group and FN100/32 kPa group, YAP/TAZ mainly localized in nuclei, while in both FN100/0.5 kPa group and FN30/32 kPa group, YAP/TAZ mainly localized in cytoplasm.
Finally, increase of matrix stiffness promoted YAP/TAZ nuclear translocation of BMSCs when cell morphology could simultaneously adapt. As shown in Fig 5B, on substrates coated with 100 µg/ml of FN, increase of matrix stiffness from 0.5 kPa to 32 kPa significantly promoted YAP/TAZ nuclear translocation.