AbstractObjectivesSecretomes of mesenchymal stromal cells (MSC) represent a novel strategy for growth‐factor delivery for tissue regeneration. The objective of this study was to compare the efficacy of adjunctive use of conditioned media of bone‐marrow MSC (MSC‐CM) with collagen barrier membranes vs. adjunctive use of conditioned media of leukocyte‐ and platelet‐rich fibrin (PRF‐CM), a current growth‐factor therapy, for guided bone regeneration (GBR).MethodsMSC‐CM and PRF‐CM prepared from healthy human donors were subjected to proteomic analysis using mass spectrometry and multiplex immunoassay. Collagen membranes functionalized with MSC‐CM or PRF‐CM were applied on critical‐size rat calvaria defects and new bone formation was assessed via three‐dimensional (3D) micro‐CT analysis of total defect volume (2 and 4 weeks) and 2D histomorphometric analysis of central defect regions (4 weeks).ResultsWhile both MSC‐CM and PRF‐CM revealed several bone‐related proteins, differentially expressed proteins, especially extracellular matrix components, were increased in MSC‐CM. In rat calvaria defects, micro‐CT revealed greater total bone coverage in the MSC‐CM group after 2 and 4 weeks. Histologically, both groups showed a combination of regular new bone and ‘hybrid’ new bone, which was formed within the membrane compartment and characterized by incorporation of mineralized collagen fibers. Histomorphometry in central defect sections revealed greater hybrid bone area in the MSC‐CM group, while the total new bone area was similar between groups.ConclusionBased on the in vitro and in vivo investigations herein, functionalization of membranes with MSC‐CM represents a promising strategy to enhance GBR.