Exposure of periodontal ligament progenitor cells to lipopolysaccharide from Escherichia coli changes osteoblast differentiation pattern

Abstract

Periodontal ligament mesenchymal stem cells (PDLMSCs) are an important alternative source of adult stem cells and may be applied for periodontal tissue regeneration, neuroregenerative medicine, and heart valve tissue engineering. However, little is known about the impact of bacterial toxins on the biological properties of PDLSMSCs, including self-renewal, differentiation, and synthesis of extracellular matrix. Objective: This study investigated whether proliferation, expression of pro-inflammatory cytokines, and osteogenic differentiation of CD105-enriched PDL progenitor cell populations (PDL-CD105+ cells) would be affected by exposure to bacterial lipopolysaccharide from Escherichia coli (EcLPS).Material and Methods : Toll-like receptor 4 (TLR4) expression was assessed in PDL-CD105+ cells by the immunostaining technique and confirmed using Western blotting assay. Afterwards, these cells were exposed to EcLPS, and the following assays were carried out: (i) cell viability using MTS; (ii) expression of the interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) genes; (iii) osteoblast differentiation assessed by mineralization in vitro, and by mRNA levels of run-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) determined by quantitative PCR.Results : PDL-CD105+ cells were identified as positive for TLR4. EcLPS did not affect cell viability, but induced a significant increase of transcripts for IL-6 and IL-8. Under osteogenic condition, PDL-CD105+ cells exposed to EcLPS presented an increase of mineralized matrix deposition and higher RUNX2 and ALP mRNA levels when compared to the control group.Conclusions : These results provide evidence that CD105-enriched PDL progenitor cells are able to adapt to continuous Escherichia coli endotoxin challenge, leading to an upregulation of osteogenic activities.

Osteogenic potential of periodontal ligament stem cells are unaffected after exposure to lipopolysaccharides

“…Furthermore, a previous study published by our research group reported the expression of TLR4 in these same cell populations. 13 To our knowledge, this is the first study to describe the expression of these receptors by PDL CD105 + progenitor cells, although previous studies have reported the expression of TLR4 in cementoblasts, 15 and of TLR2 and TLR4 in the heterogeneous PDL cell population. 9,21 To evaluate the cytotoxicity effect of PgLPS, PDLSCs were initially cultured in the presence of different concentrations of this bacterial toxin.…”

“…13 At time points of 1, 3, 7, and 10 days, cells were washed with phosphate buffered saline (GIBCO BRL). Then, 20 µL of CellTiter96 ® AQ ueous One Solution Reagent -MTS assay (PromegaCo., Ltd. Madison, USA) was added to each well and cells were incubated for 2 hours.…”

“…In parallel, in vitro mineral nodule formation was assessed at day 21 using the Alizarin Red staining (AR-S, Sigma). 13 …”

“…Samples were prepared as described previously (13) . Primers sequences for ALP, OCN, RUNX2, IL-1β, TNF-α, OCT-4 and glyceral-dehyde-3-phosphate dehydrogenase (GAPDH) are shown in Table 1.…”

Ligament-Derived Stem Cells: Identification, Characterisation, and Therapeutic Application

“…Ageing and injury/degeneration have both been shown to have an effect on the function and phenotype of various stem cell types, including MSCs [30, 31], TDSCs [32–34], and LDSCs isolated from periodontal ligament [35, 36]. However, little is known about the effects of these factors on LDSCs isolated from ACL or other ligament types.…”