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Objective: The aim of this study was to produce and characterize triple-layered cell sheet constructs with varying cell compositions combined or not with the fibrin membrane scaffold obtained by the technology of Plasma Rich in Growth Factors (mPRGF)., Materials and Methods: Human primary cultures of periodontal ligament stem cells (hPDLSCs) were isolated, and their stemness nature was evaluated. Three types of triple-layered composite constructs were generated, composed solely of hPDLSCs or combined with human umbilical vein endothelial cells (HUVECs), either as a sandwiched endothelial layer or as coculture sheets of both cell phenotypes. These three triple-layered constructs were also manufactured using mPRGF as cell sheets' support. Necrosis, glucose consumption, secretion of extracellular matrix proteins and synthesis of proangiogenic factors were determined. Histological evaluations and proteomic analyses were also performed., Results: The inclusion of HUVECs did not clearly improve the properties of the multilayered constructs and yet hindered their optimal conformation. The presence of mPRGF prevented the shrinkage of cell sheets, stimulated the metabolic activity and increased the matrix synthesis. At the proteome level, mPRGF conferred a dramatic advantage to the hPDLSC constructs in their ability to provide a suitable environment for tissue regeneration by inducing the expression of proteins necessary for bone morphogenesis and cellular proliferation., Conclusions: hPDLSCs' triple-layer construct onto mPRGF emerges as the optimal structure for its use in regenerative therapeutics., Clinical Relevance: These results suggest the suitability of mPRGF as a promising tool to support cell sheet formation by improving their handling and biological functions., Competing Interests: Competing interest The authors declare the following competing financial interests: E.A. is the Scientific Director of and M.T, M.Z., R.T., and M.H.A. are scientists at BTI Biotechnology Institute, a dental implant company that investigates in the fields of oral implantology and PRGF-Endoret technology. N.O., A.F. and A.M.Z. declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)