Abstract
ObjectivesConditioned medium (CM) from 2D cell culture can mitigate the weakened regenerative capacity of the implanted stem cells. However, the capacity of 3D CM to prime dental pulp stem cells (DPSCs) for pulp regeneration and its protein profile are still elusive. We aim to investigate the protein profile of CM derived from 3D tooth germs, and to unveil its potential for DPSCs‐based pulp regeneration.Materials and MethodsWe prepared CM of 3D ex vivo cultured tooth germ organs (3D TGO‐CM) and CM of 2D cultured tooth germ cells (2D TGC‐CM) and applied them to prime DPSCs. Influences on cell behaviours and protein profiles of CMs were compared. In vivo pulp regeneration of CMs‐primed DPSCs was explored using a tooth root fragment model on nude mice.ResultsTGO‐CM enhanced DPSCs proliferation, migration, in vitro mineralization, odontogenic differentiation, and angiogenesis performances. The TGO‐CM group generated superior pulp structures, more odontogenic cells attachment, and enhanced vasculature at 4 weeks post‐surgery, compared with the TGC‐CM group. Secretome analysis revealed that TGO‐CM contained more odontogenic and angiogenic growth factors and fewer pro‐inflammatory cytokines. Mechanisms leading to the differential CM profiles may be attributed to the cytokine–cytokine receptor interaction and PI3K‐Akt signalling pathway.ConclusionsThe unique secretome profile of 3D TGO‐CM made it a successful priming cocktail to enhance DPSCs‐based early pulp regeneration.
Highlights
IntroductionDental pulp stem cells (DPSCs) offer practical advantages such as accessibility, self-renewal, multipotency, and immunomodulation,[1] making
In vitro mesenchymal stem cells (MSC) priming is a promising technique in circumventing these shortcomings by modulating the secretome,[11] with hypoxia, cytokines, and 3D cell culture proposed as in vitro priming stimuli.12–15 Conditioned medium (CM), the supernatant collected from the cell culture medium, contains the MSC secretome, which includes extracellular matrix components, growth factors, and cytokines.[16]
3D tooth germ organs (TGOs)-CM priming achieved superior early in vivo pulp regeneration results, with well-organized pulp structures, odontogenic cell layer attachment, and increased vasculature formation at 4 weeks post-surgery. 3D TGO-CM contained more growth factors related to odontogenesis and angiogenesis and fewer pro- inflammatory cytokines
Summary
Dental pulp stem cells (DPSCs) offer practical advantages such as accessibility, self-renewal, multipotency, and immunomodulation,[1] making. In vitro MSC priming is a promising technique in circumventing these shortcomings by modulating the secretome,[11] with hypoxia, cytokines, and 3D cell culture proposed as in vitro priming stimuli.12–15 Conditioned medium (CM), the supernatant collected from the cell culture medium, contains the MSC secretome, which includes extracellular matrix components, growth factors, and cytokines.[16] Already being broadly used,[17] CM priming can promote tissue regeneration, angiogenesis, immunomodulation, and anti-fibrosis.[18] DPSC-derived CMs can recapitulate parent MSC functions, suggesting potential in dental and extra-oral tissue engineering applications.[19]
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