Toll-like receptor and C-type lectin receptor agonists attenuate osteogenic differentiation in human dental pulp stem cells

Abstract Background This study aimed to investigate the effects of various toll-like receptor (TLR) and C-type lectin receptor (CLR) ligands on osteogenic differentiation in human dental pulp stem cells (hDPSCs). Methods hDPSCs were cultured and treated with various concentrations (0.01, 0.1, 1.0, and 10 µg/mL) of TLR or CLR agonists (PG-LPS, E.coli LPS, poly(I:C), Pam3CSK4, Furfurman, and Zymosan). Cell viability was determined by MTT assay. The effects of TLR and CLR agonists on osteogenic differentiation of hDPSCs were measured by alkaline phosphatase (ALP) activity, Alizarin Red S staining, and Von Kossa staining. In addition, the mRNA expression of osteogenesis-related genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP1) was examined by RT-qPCR. A non-parametric analysis was employed for the statistical analyses. The statistically significant difference was considered when p < 0.05. Results Treatment with TLR and CLR agonists was associated with an increase in hDPSCs’ colony-forming unit ability. Compared with the control group, TLR and CLR agonists significantly inhibited the osteogenic differentiation of hDPSCs by decreasing the ALP activity, mineralised nodule formation, and mRNA expression levels of osteogenesis-related genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP1). The inhibition of TRIF but not Akt signalling rescued the effects of TLR and CLR agonist attenuating hDPSCs’ mineralisation. Conclusions The activation of TLRs or CLRs exhibited an inhibitory effect on osteogenic differentiation of hDPSCs via the TRIF-dependent signalling pathway

Functional consequences of C-terminal mutations in RUNX2

Abstract Cleidocranial dysplasia (CCD) is a genetic disorder caused by mutations in the RUNX2 gene, affecting bone and teeth development. Previous studies focused on mutations in the RUNX2 RHD domain, with limited investigation of mutations in the C-terminal domain. This study aimed to investigate the functional consequences of C-terminal mutations in RUNX2. Eight mutations were analyzed, and their effects on transactivation activity, protein expression, subcellular localization, and osteogenic potential were studied. Truncating mutations in the PST region and a missense mutation in the NMTS region resulted in increased transactivation activity, while missense mutations in the PST showed activity comparable to the control. Truncating mutations produced truncated proteins, while missense mutations produced normal-sized proteins. Mutant proteins were mislocalized, with six mutant proteins detected in both the nucleus and cytoplasm. CCD patient bone cells exhibited mislocalization of RUNX2, similar to the generated mutant. Mislocalization of RUNX2 and reduced expression of downstream genes were observed in MSCs from a CCD patient with the p.Ser247Valfs*3 mutation, leading to compromised osteogenic potential. This study provides insight into the functional consequences of C-terminal mutations in RUNX2, including reduced expression, mislocalization, and aberrant transactivation of downstream genes, contributing to the compromised osteogenic potential observed in CCD

Effects of decellularized extracellular matrix derived from Jagged1-treated human dental pulp stem cells on biological responses of stem cells isolated from apical papilla

International audienceObjective: Indirect Jagged1 immobilization efficiently activates canonical Notch signaling in human dental pulp stem cells (hDPSCs). This study aimed to investigate the characteristics of the Jagged1-treated hDPSC-derived decellularized extracellular matrix (dECM) and its biological activity on odonto/osteogenic differentiation of stem cells isolated from apical papilla (SCAPs). Methods: Bioinformatic database of Jagged1-treated hDPSCs was analyzed using NetworkAnalyst. hDPSCs seeded on the Jagged1 immobilized surface were maintained with normal or osteogenic induction medium (OM) followed by decellularization procedure, dECM-N, or dECM-OM, respectively. SCAPs were reseeded on each dECM with either the normal medium or the OM. Cell viability was determined by MTT assay. Characteristics of dECMs and SCAPs were evaluated by SEM, EDX, immunofluorescent staining, and alcian blue staining. Mineralization was assessed by alizarin red S, Von Kossa, and alkaline phosphatase staining. Statistical significance was considered at p &lt; 0.05. Results: RNA-seq database revealed upregulation of several genes involved in ECM organization, ECM–receptor interaction, and focal adhesion in Jagged1-treated hDPSCs. Immobilized Jagged1 increased the osteogenesis of the hDPSC culture with OM. dECMs showed fibrillar-like network structure and maintained major ECM proteins, fibronectin, type I-collagen, and glycosaminoglycans. A decrease in calcium and phosphate components was observed in dECMs after the decellularized process. Cell viability on dECMs did not alter by 7 days. Cell attachment and f-actin cytoskeletal organization of SCAPs proliferated on Jagged1-treated dECMs were comparable to those of the control dECMs. SCAPs exhibited significantly higher mineralization on dECM-N in OM and markedly enhanced on dECM-OM with normal medium or OM conditions. Conclusion: Jagged1-treated hDPSC-derived dECMs are biocompatible and increase odonto/osteogenic differentiation of SCAPs. The results suggested the potential of Jagged1 dECMs, which could be further developed into ECM scaffolds for application in regenerative medicine

Alteration of extracellular matrix proteins in atrophic periodontal ligament of hypofunctional rat molars

Abstract Objectives The aim of this study was to investigate the effect of mechanical force on possible dynamic changes of the matrix proteins deposition in the PDL upon in vitro mechanical and in vivo occlusal forces in a rat model with hypofunctional conditions. Materials and methods Intermittent compressive force (ICF) and shear force (SF) were applied to human periodontal ligament stem cells (PDLSCs). Protein expression of collagen I and POSTN was analyzed by western blot technique. To establish an in vivo model, rat maxillary molars were extracted to facilitate hypofunction of the periodontal ligament (PDL) tissue of the opposing mandibular molar. The mandibles were collected after 4-, 8-, and 12-weeks post-extraction and used for micro-CT and immunohistochemical analysis. Results ICF and SF increased the synthesis of POSTN by human PDLSCs. Histological changes in the hypofunctional teeth revealed a narrowing of the PDL space, along with a decreased amount of collagen I, POSTN, and laminin in perivascular structures compared to the functional contralateral molars. Conclusion Our results revealed that loss of occlusal force disrupts deposition of some major matrix proteins in the PDL, underscoring the relevance of mechanical forces in maintaining periodontal tissue homeostasis by modulating ECM composition