Mile Eraković

Background/Objectives: Photobiomodulation (PBM) has been proposed to regulate periodontal tissue remodeling, but its effects on osteoblastogenesis in periodontal ligament stem cells (PDLSCs) remain highly dependent on laser parameters. This study investigated the fluence-dependent effects of 940 nm diode laser irradiation on the proliferation and osteoblastic differentiation of human PDLSCs. Methods: Six primary human PDLSC cultures were established and characterized using flow cytometry and multilineage differentiation assays. Cell proliferation was assessed after a single irradiation at fluences of 1, 2, 4, and 10 J/cm². Osteoblastogenesis was induced using repeated irradiation regimens initiated at 2, 4, or 10 J/cm² under suboptimal or complete osteogenic conditions and evaluated by Alizarin Red staining on day 21. The expression of osteogenic genes (RUNX2, BMP2, SPP1, TGFB1, COL1A1, ALPL, and BGLAP) was evaluated by qRT-PCR at different time points. Culture supernatants were analyzed for soluble osteogenic mediators. Results: The established PDLSC cultures had an MSC-like phenotype, robust osteogenic potential, moderate chondrogenic potential, and weak or no adipogenic potential. Proliferation of PDLSCs was considerably increased by PBM at 2 J/cm² on day 7, whereas fluences of 4 and 10 J/cm² inhibited proliferation. Osteoblastic mineralization was increased with the 2 J/cm² irradiation regimen but was markedly reduced with the 10 J/cm² regimen. Gene expression analysis confirmed this fluence-dependent response under optimal culture conditions. The 2 J/cm² irradiation regimen significantly upregulated each analyzed osteogenic gene at one or more time points, whereas the 10 J/cm² regimen downregulated RUNX2, BMP2, SPP1, COL1A1, and BGLAP. PBM at 2 J/cm² also increased production of soluble BMP-2, OPG, PDGF-BB, ALP, and Leptin in a time-dependent manner. Conclusions: PBM exerts a fluence-dependent effect on human PDLSCs. A single irradiation at 2 J/cm² increased proliferation, whereas the irradiation regimen initiated at 2 J/cm² promoted osteoblastogenesis. In contrast, higher-fluence conditions, particularly the regimen initiated at 10 J/cm², were inhibitory.

Background/Objectives: Photobiomodulation (PBM) has been proposed to regulate periodontal tissue remodeling, but its effects on osteoblastogenesis in periodontal ligament stem cells (PDLSCs) remain highly dependent on laser parameters. This study investigated whether irradiation with a 940-nm diode laser elicits a biphasic dose-response in proliferation and osteoblastic differentiation assays of human PDLSCs. Methods: Six human PDLSC lines were established and characterized using flow cytometry and multilineage differentiation assays. Cell proliferation was assessed after irradiation with fluences of 1, 2, 4, and 10 J/cm². Osteoblastogenesis was induced under suboptimal or complete osteogenic conditions and evaluated by Alizarin Red staining on day 21. The expression of osteogenic genes (RUNX2, BMP2, SPP1, TGFB1, COL1A1, ALPL, and BGLAP) was evaluated by qRT-PCR at different time points. Culture supernatants were analyzed for soluble osteogenic mediators. Results: The established PDLSC lines had an MSC-like phenotype, robust osteogenic potential, moderate chondrogenic potential, and weak or no adipogenic potential. Proliferation of PDLSCs was considerably increased by PBM at 2 J/cm² on day 7, whereas fluences of 4 and 10 J/cm² inhibited proliferation. Osteoblastic mineralization was increased at 2 J/cm² but was markedly reduced at 10 J/cm². Gene expression analysis confirmed this biphasic response under optimal culture conditions. Irradiation with 2 J/cm² upregulated all the analyzed genes, whereas 10 J/cm² downregulated RUNX2, BMP2, SPP1, COL1A1, and BGLAP. PBM at 2 J/cm² also increased production of soluble BMP-2, OPG, PDGF-BB, ALP, and Leptin in a time-dependent manner. Conclusions: PBM exerts a biphasic effect on human PDLSCs that critically depends on PBM fluency. A lower energy density of 2 J/cm² promotes proliferation and osteoblastogenesis, whereas higher energy densities, particularly 10 J/cm², are inhibitory.

Biodentine, a tricalcium silicate cement, has emerged as a retrograde root-end filling material to promote periapical lesion (PL) healing after apicoectomy. However, its underlying mechanisms remain unclear. This study tested the hypothesis that Biodentine stimulates the osteogenic differentiation of mesenchymal stromal cells (MSCs) derived from PLs. The Biodentine extract (B-Ex) was prepared by incubating polymerized Biodentine in RPMI medium (0.2 g/mL) for three days at 37 °C. B-Ex, containing both released microparticles and soluble components, was incubated with PL-MSCs cultured in either a basal MSC medium or suboptimal osteogenic medium. Osteogenic differentiation was assessed by Alizarin Red staining and the expression of 20 osteoblastogenesis-related genes. Non-cytotoxic concentrations of B-Ex stimulated the proliferation of PL-MSCs and induced their osteogenic differentiation in a dose-dependent manner, with a significantly enhanced effect in suboptimal osteogenic medium. B-Ex upregulated most early and late osteoblastic genes. However, the differentiation process was prolonged, as indicated by the delayed expression of wingless-type MMTV integration site family member 2 (WNT2), bone gamma-carboxyglutamate protein (BGLAP), bone morphogenic protein-2 (BMP-2), growth hormone receptor (GHR), and FOS-like 2, AP-1 transcription factor subunit (FOSL2), compared with their expression under optimal osteogenic conditions. The stimulatory effect of B-Ex was primarily calcium dependent, as it was reduced by 85% when B-Ex was treated with the calcium-chelating agent EGTA. In conclusion, Biodentine promotes the osteogenic differentiation of PL-MSCs in a calcium-dependent manner, supporting its stimulatory role in periapical healing.

Biodentine, a tricalcium silicate cement, has emerged as a retrograde root-end filling material to promote periapical lesion (PL) healing after apicoectomy. However, its underlying mechanisms remain unclear. This study tested the hypothesis that Biodentine stimulates the osteogenic differentiation of mesenchymal stromal cells (MSCs) derived from PLs. The Biodentine extract (B-Ex) was prepared following ISO 10993-12 by incubating polymerized Biodentine in RPMI medium (0.2 g/mL) for three days at 37 °C. B-Ex, containing both released microparticles and soluble components, was incubated with PL-MSCs cultured in either basal MSC medium or suboptimal osteogenic medium. Osteogenic differentiation was assessed by Alizarin Red staining and the expression of 20 osteoblastogenesis-related genes, including transcription factors, signaling molecules, extracellular matrix proteins, bone hormones, cytokines/cytokine receptors, and bone remodeling factors. Non-cytotoxic concentrations of B-Ex stimulated the proliferation of PL-MSCs and induced their osteogenic differentiation in a dose-dependent manner, with a significantly enhanced effect in suboptimal osteogenic medium. The differentiation process was prolonged relative to optimal osteogenic conditions. The stimulatory effect of B-Ex was primarily calcium-dependent, as it was reduced by 85% when B-Ex was treated with the calcium-chelating agent EGTA. In conclusion, Biodentine promotes the osteogenic differentiation of PL-MSCs in a calcium-dependent manner, supporting its stimulatory role in periapical healing.

Pomegranate has shown a favorable effect on gingivitis/periodontitis, but the mechanisms involved are poorly understood. The aim of this study was to test the effect of pomegranate peel extract (PoPEx) on gingiva-derived mesenchymal stromal cells (GMSCs) under physiological and inflammatory conditions. GMSC lines from healthy (H) and periodontitis (P) gingiva (n = 3 of each) were established. The lines were treated with two non-toxic concentrations of PoPEX (low—10; high—40 µg/mL), with or without additional lipopolysaccharide (LPS) stimulation. Twenty-four genes in GMSCs involved in different functions were examined using real-time polymerase chain reaction (RT-PCR). PoPEx (mostly at higher concentrations) inhibited the basal expression of IL-6, MCP-1, GRO-α, RANTES, IP-10, HIF-1α, SDF-1, and HGF but increased the expression of IL-8, TLR3, TGF-β, TGF-β/LAP ratio, IDO-1, and IGFB4 genes in H-GMSCs. PoPEx increased IL-6, RANTES, MMP3, and BMP2 but inhibited TLR2 and GRO-α gene expression in P-GMSCs. LPS upregulated genes for proinflammatory cytokines and chemokines, tissue regeneration/repair (MMP3, IGFBP4, HGF), and immunomodulation (IP-10, RANTES, IDO-1, TLR3, COX-2), more strongly in P-GMSCs. PoPEx also potentiated most genes’ expression in LPS-stimulated P-GMSCs, including upregulation of osteoblastic genes (RUNX2, BMP2, COL1A1, and OPG), simultaneously inhibiting cell proliferation. In conclusion, the modulatory effects of PoPEx on gene expression in GMSCs are complex and dependent on applied concentrations, GMSC type, and LPS stimulation. Generally, the effect is more pronounced in inflammation-simulating conditions.

Gingiva-Derived Mesenchymal Stromal Cells (GMSCs) have been shown to play an important role in periodontitis. However, how P. gingivalis, one of the key etiological agents of the disease, affects healthy (H)- and periodontitis (P)-GMSCs is unknown. To address this problem, we established 10 H-GMSC and 12 P-GMSC lines. No significant differences in morphology, differentiation into chondroblasts and adipocytes, expression of characteristic MSCS markers, including pericyte antigens NG2 and PDGFR, were observed between H- and P-GMSC lines. However, proliferation, cell size and osteogenic potential were higher in P-GMSCs, in contrast to their lower ability to suppress mononuclear cell proliferation. P. gingivalis up-regulated the mRNA expression of IL-6, IL-8, MCP-1, GRO-α, RANTES, TLR-2, HIF-1α, OPG, MMP-3, SDF-1, HGF and IP-10 in P-GMSCs, whereas only IL-6, MCP-1 and GRO-α were up-regulated in H-GMSCs. The expression of MCP-1, RANTES, IP-10 and HGF was significantly higher in P-GMSCs compared to H-GMSCs, but IDO1 was lower. No significant changes in the expression of TLR-3, TLR-4, TGF-β, LAP, IGFBP4 and TIMP-1 were observed in both types of GMSCs. In conclusion, our results suggest that P-GMSCs retain their pro-inflammatory properties in culture, exhibit lower immunosuppressive potential than their healthy counterparts, and impaired regeneration-associated gene induction in culture. All these functions are potentiated significantly by P. gingivalis treatment.