Acid Dentin Lysate Modulates Macrophage Polarization and Osteoclastogenesis In Vitro

Abstract

Dentin prepared from extracted teeth is used as autograft for alveolar bone augmentation. Graft consolidation involves the acid lysis of dentin thereby generating a characteristic paracrine environment. Acid lysate of dentin is mimicking this environment. Acid dentin lysate (ADL) potentially targets hematopoietic cells thereby affecting their differentiation towards macrophages and osteoclasts; however, the question remains if ADL controls macrophage polarization and osteoclastogenesis. Here, we show that ADL reduced lipopolysaccharide (LPS)-induced macrophage polarization of the pro-inflammatory (M1) phenotype, indicated by attenuated Interleukin 1 (IL1), Interleukine 6 (IL6)and cyclooxygenase 2 (COX2) expression. This decrease in M1 macrophages was confirmed by the reduced phosphorylation and nuclear translocation of p65 in the LPS-exposed RAW 264.7 macrophages. Similarly, when RAW 264.7 macrophages were incubated with other agonists of Toll-like receptor (TLR) signaling e.g., FSL1, Polyinosinic-polycytidylic acid High Molecular Weight (Poly (1:C) HMW), Pam3CSK4, and imiquimod, ADL reduced the IL6 expression. We further show herein that ADL decreased osteoclastogenesis indicated by the reduced formation of multinucleated cell expressing cathepsin K and tartrate-resistant acid phosphatase in murine bone marrow cultures. Overall, our results suggest that acid dentin lysate can affect the differentiation of hematopoietic cells to M1 macrophage polarization and a decrease in osteoclastogenesis in bone marrow cultures.

Bone Allograft Acid Lysates Change the Genetic Signature of Gingival Fibroblasts

“…In accordance with our previous research [17,18,[28][29][30][31], blocking TGF-β receptor type I kinase with the inhibitor SB431542 reduced the expression of IL11, similar to what we have observed with AREG and C11orf96. However, we cannot blame allograft-derived TGF-β for mediating the increase in IL11, AREG, and C11orf96-first, we could not identify TGF-β by immunoassay and proteomics, and second, ALBA failed to provoke the activation of the canonical TGF-β signaling pathway culminating in the nuclear translocation of smad2/3.…”

“…The bioassay approach presented here considers the experience we have gained from analyzing acid bone [17,18,28] and dentine lysates [29][30][31] as well as bone-conditioned medium [32], overall showing that IL11 is a sensitive gene that is strongly regulated in gingival fibroblasts exposed to lysates and conditioned medium. IL11 is presumably more than just a biomarker as IL11 plays a central role in mediating the fibrotic activity of TGFβ [19,20], for instance, in cardiovascular and pulmonary fibrosis [33,34] as well as systemic sclerosis [35], non-alcoholic steatohepatitis [36], and kidney dysfunction [37].…”

“…Indeed, the pH the osteoclasts use to demineralize the bone is around pH = 4.5 [58], hence it was by far too high to simulate the physiological scenario. Considering this limitation, 1.0 M HCl was used to demineralize the dentine matrix based on previous bioassays [29] and, in vitro, TGF-β is activated by low pH, lower than the osteoclasts provide [59]. Nevertheless, we are indeed at risk of losing the activity of other potential bioactive molecules that are in ALBA-however, in the proteomics analysis, which is independent of the low pH, we could not consistently identify growth factors and we were not able to detect TGF-β.…”