Aim: The present study aimed to find bone substitutes to enhance osteogenic differentiation of mesenchymal stem cells in three-dimensional scaffolds in the absence of dexamethasone. Materials and Methods: Seven commercial bone substitutes were added to a three-dimensional fibrin-matrix containing rat mesenchymal stem cells in a biocompatible poly-L-lactic-acid mesh. Cell viability, cytotoxicity and alkaline phosphatase activity were followed for three weeks. Expression of bone markers was examined by qualitative evaluation of corresponding transcripts. Results: Six out of the seven bone derivatives exhibited an osteogenic-enhancing effect. Conclusion: The osteogenic-enhancing effect of the evaluated bone substitutes suggests their potential clinical application for preparation of autologous bone replacement material in three-dimensional carriers.