Response of human peripheral blood monocyte-derived macrophages (PBMM) to demineralized and decellularized bovine bone graft substitutes

Abstract

The performance of apparently biocompatible implanted bovine bone grafts may be compromised by unresolved chronic inflammation, and poor graft incorporation leading to implant failure. Monitoring the intensity and duration of the inflammatory response caused by implanted bone grafts is crucial. In this study, the ability of demineralized (DMB) and decellularized (DCC) bovine bone substitutes in initiating inflammatory responses to peripheral blood monocyte-derived macrophages (PBMMs) was investigated. The response of PBMMs to bone substitutes was evaluated by using both direct and indirect cell culture, reactive oxygen species (ROS) generation, apoptosis, immunophenotyping, and cytokine production. Analysis of DMB and DCC substitutes using scanning electron microscope (SEM) showed a roughened surface with a size ranging between 500 and 750 μm. PBMMs treated with DMB demonstrated cell aggregation and clumping mimicking lipopolysaccharide (LPS) treated PBMMs and a higher proliferation ability (166.93%) compared to control (100%) and DCC treatments (115.64%; p<0.001) at 24h. This was associated with a significantly increased production of intracellular ROS in PBMMs exposed to DMB substitutes than control (3158.5 vs 1715.5; p<0.001) and DCC treatment (2117.5). The bone substitute exposure also caused an increase in percentage apoptosis which was significantly (p<0.0001) higher in both DMB (27.85) and DCC (29.2) treatment than control (19.383). A significant increase in proinflammatory cytokine expression (TNF-α: 3.4 folds; p<0.05) was observed in DMB substitute-treated PBMMs compared to control. Notably, IL-1β mRNA was significantly higher in DMB (21.75 folds; p<0.0001) than control and DCC (5.01 folds). In contrast, DCC substitutes exhibited immunoregulatory effects on PBMMs, as indicated by the expression for CD86, CD206, and HLDR surface markers mimicking IL-4 treatments. In conclusion, DMB excites a higher immunological response compared to DCC suggesting decellularization process of tissues dampen down inflammatory reactions when exposed to PBMM.

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.