Table 1.
Primers used in qPCR.
Fig 1.
Scanning Electron Microscope image analysis of (a) demineralized bone (DMB) and (b) decellularized bone (DCC) substitutes.
The DMB and DCC substitutes ranged in size from 500–750μm. Scale bar = 400 μm.
Fig 2.
Morphology of peripheral blood monocyte-derived macrophages (PBMM) exposed to DMB and DCC.
Representative images showing the morphology of PBMMs grown in (a-e) direct and (f, g) indirect presence of DMB and DCC substitutes in comparison to untreated, LPS (b) and IL-4 treated cells (c) on day 8 of culture. Arrows indicate proliferation and clumping of cells (b, d & f). Arrow heads indicate DCC treated PBMMs exhibiting a spread-out morphology similar to IL-4 treatments. (c, e & g). Scale bar = 50 μm.
Fig 3.
Cell proliferation assay of PBMCs grown in direct (a) or indirect (b) contact with DCC and DMB substitutes in comparison to LPS (100ng/mL), IL-4 (15ng/mL) and cells grown in normal tissue culture medium.
*p<0.01, **p<0.001, and ns—non-significant is indicated. Error bars represent standard error of means of three independent measurements.
Fig 4.
Reactive oxygen species (ROS) generation and cellular apoptosis in PBMMs post-exposure to DMB and DCC substitutes in comparison to untreated control cells.
(a) A significant increase in ROS generation was observed for cells grown in the presence of DMB substitutes at day 1 and day 8 when compared to control cells (***p < 0.001). (b) FITC-Annexin V and propidium iodide (PI) cell apoptotic assay of PBMMs. Flow cytometry dot plots showing cellular apoptosis in PBMMs grown for day 1 and day 8 in the presence of 1mg/mL of DMB and DCC substitutes in comparison to control cells grown in normal tissue culture medium. (c) Percentage of both early (Q3) and late (Q2) apoptosis in PBMMs. ****p < 0.0001, and ns–no significance is indicated. Error bars represent standard error of means of three independent experiments.
Fig 5.
Phenotypic analysis of PBMMs polarization after treatment with LPS, IL-4, DMB and DCC substitutes for 8 days.
Phenotypic characterization of macrophages after treatment with bone substitutes was investigated by examining the differential expression of macrophage-related markers like CD14, CD16, CD86, and CD206 based on MFI on gated events. Phenotypic characterization of macrophages after treatment with bone substitutes was investigated by examining the differential expression of macrophage-related markers like CD14, CD16, CD86, and CD206 based on MFI acquired from gated events. One-way ANOVA was used to analyze the data, and the findings were presented as mean + standard error of means. * p<0.05, **< p0.01, *** p<0.001, and **** p<0.0001 is indicated. Error bars represent standard error of means of three independent experiments.
Fig 6.
The expression of TNF-α, IL1-β, and IL-10 cytokines and CD80 and CD206 markers in PBMMs treated with 1mg/mL of DMB or DCC substitutes in comparison to LPS and IL-4 treatments, or untreated control cells.
The data was normalized with cells grown in a normal tissue culture medium and GAPDH served as the internal housekeeping control gene. *p<0.05, and ****p<0.0001 and ns-non-significant are indicated. Error bars represent standard error of means of three independent experiments.
Table 2.
Summary of cellular characteristics and responses in PBMMs treated with DMB and DCC substitutes.