TLR2 and TLR4 Differentially Regulate the Osteogenic Capacity of Human Periodontal Ligament Fibroblasts

To test that the osteogenic capacity of periodontal ligament (PDL) fibroblasts can be mediated by TLR2 and TLR4 activation.

Human PDL fibroblasts were cultured in osteogenic medium and treated with TLR2 and TLR4 agonists (Pam3CSK4 and monophosphoryl Lipid A (MPLA), respectively). Cell proliferation was measured by MTT and BrdU incorporation. Osteogenic differentiation was measured by alkaline phosphatase (ALP) activity. Nodule formation was measured for osteoblast function. The expression of markers of potential signaling pathways (RUNX2, OCN, BSP and Osterix) was evaluated by quantitative PCR.

PDL fibroblasts grew at the same rate during the first 5 days in response to both Pam3CSK5 and MPLA. On day 7, cells cultured in the presence of Pam3CSK4 had a significantly higher rate of DNA replication, while cells in MPLA group had a significantly lower DNA replication rate (one-third) compared to the control (p less than 0.05). Pam3CSK4 induced significantly higher ALP activity and larger calcified nodules. TLR4 activation significantly reduced the expression of RUNX2 and osterix and enhanced OCN. Neither TLR2 nor TLR4 affected BSP expression.

These data suggest that the activation of TLR2 and TLR4 differentially and perhaps antagonistically modulate osteogenesis by human PDL fibroblasts and have a direct role of TLR-mediated PDL function during periodontal regeneration as a potential target for therapeutics.