The dose–effect relationship between the seeding quantity of human marrow mesenchymal stem cells and in vivo tissue-engineered bone yield

Abstract

Although the feasibility of human bone marrow mesenchymal stem cell (hBMMSC)-based tissue-engineered bone (TEB) has been proven in a number of studies, reaching a high positive fraction and bone yield of TEB still remains a challenge. Here we report a dose–effect relationship of the quantity of seeded cells with in vivo bone yield and the required quantity of hBMMSCs for the effective, stable bone formation of TEB. In our study, TEB was constructed using the static seeding technique with the gradient of seeding densities and volumes of passage 3 hBMMSCs. The in vitro characteristics of seeding efficiency, proliferation, viability, distribution, and osteogenic differentiation of hBMMSCs seeded on two commercial scaffolds of b-TCP and CHA were investigated using alamarBlue assay, live/dead staining, confocal laser scanning microscope, scanning electronic microscopy examination, and mRNA expression analysis of osteogenic differentiation markers. After 3 months of ectopic implantation, in vivo bone regeneration was examined by quantitative analysis of histology and micro-CT. The results showed that 10×106 cells/ml was the minimum cell seeding density for CHA and b-TCP to generate new bone in vivo. In addition, 20×106 cells/ml and 30×106 cells/ml were the saturating seeding densities for CHA and b-TCP to produce new bone effectively and stably, respectively. Thus, for different scaffolds, the saturating seeding density should be investigated first to ensure the effectiveness and stability of TEB construction with minimum donor injury, which is essential for the clinical application of TEB.