IADR Abstract Archives
Assessing the effects of Hyperglycaemia and Lipopolysaccharide on Osteogenic Differentiation
Objectives: Porphyromonas gingivalis is a key pathogen implicated in mediating periodontitis, via the release of products, such as lipopolysaccharide (Pg-LPS), which perpetuate host immuno-inflammatory reactions, hinder repair processes and promote bone tissue loss. Diabetes mellitus also influences periodontitis, as hyperglycaemia can alter cellular behaviour leading to impaired healing. Bone-marrow, derived-mesenchymal stem cells (MSCs) are essential for these repair responses, existing as heterogeneous populations that vary in their proliferative and differentiation capabilities. This study investigated the respective impact of hyperglycaemia and Pg-LPS on the osteogenic potential of MSCs.
Methods: MSCs were isolated from rat femur bone chips and cultured to specific population doubling levels (PDs); PD15, previously characterised to represent a mixed populations of immature and lineage restricted MSCs, and PD50 previously characterised to contain more immature MSCs. Cells were cultured in physiologically normal (5.5mM, NG) or high (25mM, HG) glucose levels for 28 days, in osteogenic media with/without Pg-LPS at sub-lethal concentrations (0-1µL/mL). Osteogenic differentiation was quantified by increase gene expression of osterix, osteopontin and osteocalcin using qPCR, and mineral deposition by Alizarin Red staining.
Results:
Considering results for all osteogenic markers, MSCs at PD15 demonstrated a greater response to osteogenic stimuli, compared to PD50 cells, confirming previous results that PD15 contained more committed osteoprogenitor cells. In the presence of HG, osteogenic differentiation decreased for PD15 MSCs, but the additional presence of Pg-LPS had no further detrimental effect. For PD50 cells HG exerted no effect on osteogenic markers, but additional presence of Pg-LPS inhibited osteogenic differentiation.
Conclusions:
Of these two factors, HG appears to exert a more potent effect, especially on the mixed immature/committed MSCs. Pg-LPS had a greater inhibitory effect on osteogenic differentiation by MSCs with a more immature phenotype. These results are valuable in elucidating how immature and committed MSCs respond differently to glucose and pathogenic factors contributing to periodontal tissue destruction.
Methods: MSCs were isolated from rat femur bone chips and cultured to specific population doubling levels (PDs); PD15, previously characterised to represent a mixed populations of immature and lineage restricted MSCs, and PD50 previously characterised to contain more immature MSCs. Cells were cultured in physiologically normal (5.5mM, NG) or high (25mM, HG) glucose levels for 28 days, in osteogenic media with/without Pg-LPS at sub-lethal concentrations (0-1µL/mL). Osteogenic differentiation was quantified by increase gene expression of osterix, osteopontin and osteocalcin using qPCR, and mineral deposition by Alizarin Red staining.
Results:
Considering results for all osteogenic markers, MSCs at PD15 demonstrated a greater response to osteogenic stimuli, compared to PD50 cells, confirming previous results that PD15 contained more committed osteoprogenitor cells. In the presence of HG, osteogenic differentiation decreased for PD15 MSCs, but the additional presence of Pg-LPS had no further detrimental effect. For PD50 cells HG exerted no effect on osteogenic markers, but additional presence of Pg-LPS inhibited osteogenic differentiation.
Conclusions:
Of these two factors, HG appears to exert a more potent effect, especially on the mixed immature/committed MSCs. Pg-LPS had a greater inhibitory effect on osteogenic differentiation by MSCs with a more immature phenotype. These results are valuable in elucidating how immature and committed MSCs respond differently to glucose and pathogenic factors contributing to periodontal tissue destruction.