Aims: Human periodontal ligament stem cells (hPDLSCs) exhibit an enormous potential to regenerate periodontal tissue. However, their translatability to the clinical setting is constrained by technical difficulties in standardizing culture conditions. The aim was to assess complex culture conditions using a proteomic-based protocol to standardize multi-layer hPDLSC cultivation methodology.
Materials and methods: hPDLSC-derived constructs were created with varying biological complexity. The simplest constructs were monolayer sheets of hPDLSCs cultured with fetal bovine serum (FBS) or Plasma Rich in Growth Factors supernatant (PRGFsn). The most complex constructs were triple-layered cell structures cultured with PRGFsn, with or without PRGF fibrin membrane (mPRGF). Ultrastructure and proteomic analyses were performed on these constructs.
Results: PRGF supernatant improved protein expression related to extracellular matrix, adhesion, proliferation, and migration in hPDLSCs. PRGF fibrin scaffold upregulates proteins for cell activation, respiration, and electron transport. hPDLSCs on fibrin membrane show robust osteogenic potential through differential protein expression (ossification, tissue remodeling, morphogenesis, or cell migration) and overall homeostasis relative to less complex structures.
Conclusion: Our data reveal the far-reaching potential of 3-dimensional constructs in combination with PRGF technology in periodontal regenerative applications.
Keywords: Human periodontal ligament stem cells; cell sheet technology; osteogenic differentiation; plasma rich in growth factors; proteome; triple-layered cell constructs.
What this summary about?: Human periodontal ligament stem cells (hPDLSCs) – cells that can transform into bone-like cells – could be better utilized to regenerate periodontal tissue. Although promising, it’s challenging to use these cells in clinical settings due to difficulties in standardizing how they’re cultured. To understand the effects of various cell culture methods on the cells’ protein makeup and their suitability for regenerative medicine, the researchers created different hPDLSC constructs, from simple cell sheets to more complex, multi-layered structures.What were the results?: From all culture conditions, the use of Plasma Rich in Growth Factors (PRGF), both as cell culture medium and scaffold, enhances the cells homeostasis and their ability to differentiate into bone-forming cells. Advanced imaging techniques showed good interactions between the cells and the 3D scaffold of PRGF.What do the results mean?: The findings reveal that the more complex, multi-layered constructs, especially those grown with PRGF, show better differentiation potential and stability compared to simpler structures. This suggests that combining 3D cell constructs with PRGF technology could be a powerful approach for periodontal regeneration in clinical applications.