Altered Osteogenic Differentiation in Mesenchymal Stem Cells Isolated from Compact Bone of Chicken Treated with Varying Doses of Lipopolysaccharides

Abstract

Persistent inflammation biologically alters signaling molecules and ultimately affects osteogenic differentiation, including in modern-day broilers with unique physiology. Lipopolysaccharides (LPS) are Gram-negative bacterial components that activate cells via transmembrane receptor activation and other molecules. Previous studies have shown several pathways associated with osteogenic inductive ability, but the pathway has yet to be deciphered, and data related to its dose-dependent effect are limited. Primary mesenchymal stem cells (MSCs) were isolated from the bones of day-old broiler chickens, and the current study focused on the dose-dependent variation (3.125 micrograms/mL to 50 micrograms/mL) in osteogenic differentiation and the associated biomarkers in primary MSCs. The doses in this study were determined using a cell viability (MTT) assay. The study revealed that osteogenic differentiation varied with dose, and the cells exposed to higher doses of LPS were viable but lacked differentiating ability. However, this effect became transient with lower doses, and this phenotypic character was observed with differential staining methods like Alizarin Red, Von Kossa, and alkaline phosphatase. The data from this study revealed that LPS at varying doses had a varying effect on osteogenic differentiation via several pathways acting simultaneously during bone development.

Keywords: bone development; broilers; interleukin 1 beta; lipopolysaccharides; osteogenic differentiation.

Figure 1

Illustrates the objective of the current study representing the source of LPS, along with their effect, hypothesized to establish an in vitro model for understanding inflammatory bone diseases.

Figure 2

Represents the protocol for isolating MSCs (mesenchymal stem cells) from the compact bones of chicken.

Figure 3

Represents the cell viability assay when MSCs were treated with LPS conducted at 6, 12, 24, and 48 h. GM and DM are negative and positive controls, respectively. Treatments with different letters indicate significant differences between treatments using Tukey’s HSD test, p < 0.05. Data shown include mean ± SEM of six individual replicates (n = 6).

Figure 4

Represents the osteogenic differentiation staining at day 14, which revealed the decrease in differentiation (A,C) and alkaline phosphatase activity (B) with an increase in dose of lipopolysaccharides (LPS). GM and DM are negative and positive controls, respectively.

Figure 5

Represents the ROS production in chicken MSCs at 2.5 and 4 h of treatment. Treatments with different letters indicate significant differences between treatments using Tukey’s HSD test, p < 0.05. Data shown include mean ± SEM of five individual replicates (n = 5).

Figure 6

Represents the effect of LPS on chicken MSCs in CASP8 and NRF2 gene expression at 24 h of treatment. Treatments with different letters indicate significant differences between treatments using Tukey’s HSD test, p < 0.05. Data shown include mean ± SEM of three individual replicates (n = 3).

Figure 7

Represents the effect of LPS on chicken MSCs in terms of IL-1β, TLR4, and DICER1 gene expression at 24 h of treatment (48 h treatment for IL-1β). Treatments with different letters indicate significant differences between treatments using Tukey’s HSD test, p < 0.05. Data shown include mean ± SEM of three individual replicates (n = 3).

Figure 8

Represents the effect of LPS on chicken MSCs in LRP5, CTNNB1, and SOST gene expression at 24 h of treatment. Treatments with different letters indicate significant differences between treatments using Tukey’s HSD test, p < 0.05. Data shown include mean ± SEM of three individual replicates (n = 3).

Figure 9

Represents the effect of LPS on chicken MSCs in SMAD1 and RANKL gene expression at 24 h of treatment. Treatments with different letters indicate significant differences between treatments using Tukey’s HSD test, p < 0.05. Data shown include mean ± SEM of three individual replicates (n = 3).

Figure 10

Summarizes the possible pathways affecting osteogenic differentiation in the current study at lower (3.125 μg/mL and 6.25 μg/mL) and higher (25 μg/mL and 50 μg/mL) doses.