Fullaondo Asier, Zalduendo Mar, Osinalde Nerea, Alkhraisat Mohammad H, Anitua Eduardo, Zubiaga Ana M
Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country (UPV/EHU), Leioa, Spain.
UIRMI (UPV/EHU-Fundación Eduardo Anitua), University Institute for Regenerative Medicine & Oral Implantology, Vitoria, Spain.
Regen Med. 2025 Jan;20(1):21-34. doi: 10.1080/17460751.2024.2445931. Epub 2025 Jan 4.
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.
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.
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.
Our data reveal the far-reaching potential of 3-dimensional constructs in combination with PRGF technology in periodontal regenerative applications.
人牙周膜干细胞（hPDLSCs）在牙周组织再生方面具有巨大潜力。然而，其在临床应用中的可转化性受到标准化培养条件技术难题的限制。本研究旨在使用基于蛋白质组学的方案评估复杂培养条件，以标准化多层hPDLSC培养方法。
构建具有不同生物学复杂性的hPDLSC衍生构建体。最简单的构建体是用胎牛血清（FBS）或富含生长因子的血浆上清液（PRGFsn）培养的hPDLSC单层片。最复杂的构建体是用PRGFsn培养的三层细胞结构，有或没有PRGF纤维蛋白膜（mPRGF）。对这些构建体进行超微结构和蛋白质组学分析。
PRGF上清液改善了hPDLSCs中与细胞外基质、黏附、增殖和迁移相关的蛋白质表达。PRGF纤维蛋白支架上调了细胞激活、呼吸和电子传递相关的蛋白质。相对于不太复杂的结构，纤维蛋白膜上的hPDLSCs通过差异蛋白质表达（骨化、组织重塑、形态发生或细胞迁移）和整体内环境稳定显示出强大的成骨潜力。
我们的数据揭示了三维构建体与PRGF技术相结合在牙周再生应用中的深远潜力。
