Fig 1: Increased biological activity of A549 cells after cultivation on PVA polyP coacervate, “PVA/polyP:Coa”. (I.) Different cell morphology of A549 cells cultured for 48 h on (A and C) particulate “Ca-polyP-NP” (“PVA/polyP:NP”) versus those contacted with (B and D) “PVA/polyP:Coa”. On the coacervate layer, the density of the cells is higher and the cell bodies are more compact compared to the particulate polyP in PVA. (II.) Response of A549 cells to different polyP environments. The cells were suspended on PVA layers, either on PVA control (black bars), particulate “PVA/polyP:NP” (red bars) or on “PVA/polyP:Coa” (blue bars) and incubated for 1 h or 2 h. Then the supernatants were collected and the ATP content determined using the luciferin-luciferase-based Enlighten assay. Significant pairs are marked (∗p < 0.01). In parallel series, the effect of the ADK inhibitor Ap5A (hatched) and the ALP inhibitor LEV (cross-hatched) on the ATP production of A549 cells cultured onto the three different matrices was determined as well. Significant differences are marked (& p < 0.01).
Fig 2: Role of TCONS_00072128-mediated caspase-8 activation in osteogenic differentiation of BMSCs. (A–D) Western blot analysis with caspase 8and ALP extracted from BMSCs treated with lentivirus infection. OE, overexpressed TCONS_00072128. Sh, depressed TCONS_00072128. (E) ARS staining for BMSCs treated with lentivirus infection at day 1 and day 7. All results are presented with three replicates. *p ≤ 0.05, ***p ≤ 0.001.
Fig 3: Verification of the effect of exosomes on osteogenic differentiation. (A) ALP staining for MC3T3-E1 cells treated with NEXO and FEXO, individually. (B,C) qPCR and WB analysis of osteogenesis-related gene expressions in MC3T3-E1 cells. All results are presented with three replicates. *p ≤ 0.05, ***p ≤ 0.001.
Fig 4: NP in the cytoplasm of A549 cells and distribution of ALP in A549 cells, the enzyme that hydrolyzes the polyP in NP. (I.) Uptake kinetics of polyP NP by A549 cells; TEM. (A) At time zero, no nanoparticles (NP) can be seen. (B and C) After incubation onto the “PVA/polyP:NP” layer for 2 h or 3 h, (D) the cells were transferred on a non-supplemented PVA substratum and incubated for a further 3 h. On the “PVA/polyP:Coa” layer, the cells take up NP. The NP are absent on the plain PVA surface; the darker shaded areas could represent coacervate areas (Coa?). (II.) Increased expression of ALP in A549 cells cultured on “PVA/polyP:Coa”; light microscopy. (A) The cells growing on the hydrogel without polyP (“PVA”) for 2 d show a low staining reaction and only a scattered distribution pattern compared to those cultured on (B) “PVA/polyP:NP”. (C) In cultures on “PVA/polyP:Coa”, however, the staining is significantly stronger and the density of the cells is higher. (D) Suppression of the staining reaction after addition of the ALP inhibitor LEV to cultures on “PVA/polyP:Coa”.
Fig 5: Droplet digital PCR results for osteogenic marker gene expression in DPSCs and hFOB 1.19 cells cultured on scaffolds: ALPL (alkaline phosphatase), RUNX2 (Runt-related transcription factor 2), and SPP1 (osteopontin). One-way ANOVA with Tukey’s post hoc tests for multiple comparisons was performed.
Supplier Page from Abcam for Alkaline Phosphatase Staining Kit (Purple)