IADR Abstract Archives
Comparison of Secretomes Derived from Periodontal Ligament and Bone Marrow Mesenchymal Stem Cells
Objectives: The secretome comprises a broad spectrum of molecules secreted by cells and plays a pivotal role in autocrine and paracrine cell-cell signalling and regulation of tissue responses. This study aimed at investigating the effect of culture conditions on production and bioactivity of secretomes from periodontal ligament stem cells (PDLSCs) and bone marrow mesenchymal stem cells (BMSCs) as well as the influence of timing of collection and filtration.
Methods: Rat PDLSCs and BMSCs at passages 3-4 were cultured in hypoxia (2% oxygen) or normoxia (21% oxygen) for 2 or 3 days in serum-free α-MEM media. The conditioned media (CM) were collected and centrifuged at 200g to remove cellular debris. Half of the samples were filtered (0.22µm pore) and all samples were stored in -80°c before analysis. The bioactivity of the CM was analysed for alkaline phosphatase (ALP) stimulatory activity in pre-osteoblasts (MC3T3) following treatment with the CMs. CM growth factor levels, including VEGF, TGF-β1 and IGF-1, were determined using commercially available ELISA kits.
Results: ALP activity was enhanced when pre-osteoblast cells were incubated with unfiltered CM from cells cultured under hypoxia. Hypoxic incubation resulted in significantly increased production of VEGF and TGF-β1 by both PDLSCs and BMSCs especially on day 3 of CM collection. Filtration reduced the level of TGF-β1, VEGF and IGF-1 in PDLSC CMs and TGF-β1 in BMSC CMs compared with unfiltered samples. In general, BMSCs produced significantly higher levels of VEGF and TGF-β1 compared with PDLSCs.
Conclusions: Data indicate differences in secretome composition derived from BMSCs and PDLSCs. Moreover, the different culture conditions and filtration approaches affected content of the MSC secretomes. Further work is warranted to determine detailed protein and bioactivity profiles of the different secretomes and to establish optimal, biologically effective secretomes which may be harnessed therapeutically to promote tissue repair.
Methods: Rat PDLSCs and BMSCs at passages 3-4 were cultured in hypoxia (2% oxygen) or normoxia (21% oxygen) for 2 or 3 days in serum-free α-MEM media. The conditioned media (CM) were collected and centrifuged at 200g to remove cellular debris. Half of the samples were filtered (0.22µm pore) and all samples were stored in -80°c before analysis. The bioactivity of the CM was analysed for alkaline phosphatase (ALP) stimulatory activity in pre-osteoblasts (MC3T3) following treatment with the CMs. CM growth factor levels, including VEGF, TGF-β1 and IGF-1, were determined using commercially available ELISA kits.
Results: ALP activity was enhanced when pre-osteoblast cells were incubated with unfiltered CM from cells cultured under hypoxia. Hypoxic incubation resulted in significantly increased production of VEGF and TGF-β1 by both PDLSCs and BMSCs especially on day 3 of CM collection. Filtration reduced the level of TGF-β1, VEGF and IGF-1 in PDLSC CMs and TGF-β1 in BMSC CMs compared with unfiltered samples. In general, BMSCs produced significantly higher levels of VEGF and TGF-β1 compared with PDLSCs.
Conclusions: Data indicate differences in secretome composition derived from BMSCs and PDLSCs. Moreover, the different culture conditions and filtration approaches affected content of the MSC secretomes. Further work is warranted to determine detailed protein and bioactivity profiles of the different secretomes and to establish optimal, biologically effective secretomes which may be harnessed therapeutically to promote tissue repair.