Proteomic and genomic analysis of acid dentin lysate with focus on TGF-β signaling

Abstract

Particulate autologous tooth roots are increasingly used for alveolar bone augmentation; however, the proteomic profile of acid dentin lysate and the respective cellular response have not been investigated. Here we show that TGF-β1 is among the 226 proteins of acid dentin lysate (ADL) prepared from porcine teeth. RNA sequencing identified 231 strongly regulated genes when gingival fibroblasts were exposed to ADL. Out of these genes, about one third required activation of the TGF-β receptor type I kinase including interleukin 11 (IL11) and NADPH oxidase 4 (NOX4). Reverse transcription-quantitative polymerase chain reaction and immunoassay confirmed the TGF-β-dependent expression of IL11 and NOX4. The activation of canonical TGF-β signaling by ADL was further confirmed by the phosphorylation of Smad3 and translocation of Smad2/3, using Western blot and immunofluorescence staining, respectively. Finally, we showed that TGF-β activity released from dentin by acid lysis adsorbs to titanium and collagen membranes. These findings suggest that dentin particles are a rich source of TGF-β causing a major response of gingival fibroblasts.

Acid Dentin Lysate Modulates Macrophage Polarization and Osteoclastogenesis In Vitro

“…This study extends previous findings from proteomic analysis and RNA sequencing revealing a potent dentine-derived TGF-β activity that targets the fibroblasts, cells of the mesenchymal lineage [15]. Hematopoietic lineage cells differentiating towards the macrophage and osteoclast lineage are also critically involved in bone regeneration [37,38].…”

“…According to our findings that ADL pushed an M1-to-M2 polarization switch and reduced osteoclastogenesis, we can extend our knowledge towards this cell lineage. The decrease in M1 inflammation was rather unexpected because ADL, particular when prepared from uncleaned teeth, has a pro-inflammatory activity in gingival fibroblast [15] and in RAW 264.7 cells [17]. However, in contrast to our previous work we have switched from 0.1 M HCL to 1.0 M HCl to prepare ADL.…”

“…TGFβ although highly abundant in ADL is not a candidate as recombinant TGF-β has no major impact on M1-to-M2 polarization switch and even increases osteoclastogenesis in vitro [43]. One other candidate molecule could be IL4 known to drive arginase expression and to block osteoclastogenesis, but IL4 was not detected in the proteomic analysis of ADL [15]. Periostin being present in ADL in contrast even pushes macrophage recruitment [15,44].…”

“…One in vitro approach is based on the study of acid dentin lysate (ADL) simulating the acid lysis of the bone-resorbing osteoclasts [14]. In our approach, porcine dentin is grinded and exposed to hydrochloric acid [15][16][17]. The ADL is then processed towards neutralizing the pH and achieving sterility before subjecting it to further exploration.…”

“…The ADL is then processed towards neutralizing the pH and achieving sterility before subjecting it to further exploration. Proteomic analysis of ADL revealed a variety of bioactive molecules including transforming growth factor-β1 (TGF-β1) that maintains its activity after processing [15,16]. RNA sequencing revealed the complex cellular response of mesenchymal cells when exposed to ADL [16,17] and ADL did not impair bone formation in rat calvarial defects [18].…”