Effects of Initial Cell Density and Hydrodynamic Culture on Osteogenic Activity of Tissue-Engineered Bone Grafts

Abstract

This study aimed to study the effects of initial cell density and in vitro culture method on the construction of tissue-engineered bone grafts and osteogenic activities. Human mesenchymal stem cells (hMSCs) were seeded onto cubic scaffolds prepared from demineralized bone matrix (DBM) by three methods - static, hydrodynamic, or fibrin hydrogel-assisted seeding. The resulting cell-scaffold constructs were cultured in vitro by static flask culture or hydrodynamic culture. The initial cell density and the subsequent in vitro proliferation and alkaline phosphate activities of the constructs were analyzed. The constructs were also subcutaneously implanted in nude mice to examine their in vivo osteogenic activities. Hydrogel-assisted seeding gave the highest seeding efficiency, followed by hydrodynamic and conventional static seeding. During in vitro culture, hydrodynamic culture produced higher plateau cell densities, alkaline phosphatase (ALP) activities, and extracellular matrix production than static culture. After subcutaneous implantation in nude mice, the implants prepared by the combination of hydrogel-assisted seeding and hydrodynamic culture produced higher wet weight and bone mineral density than implants prepared by other methods. The results suggest that the hydrogel-assisted seeding can substantially increase the initial seed cell density in scaffolds. Subsequent hydrodynamic culture can promote the proliferation and osteoblastic differentiation of the seeded cells. Correspondingly, bone grafts produced by the combination of these two methods achieved the highest osteogenic activity among the three methods employed.

Adhesion, proliferation and osteogenic differentiation of human MSCs cultured under perfusion with a marine oxygen carrier on an allogenic bone substitute

“…It was reported that the initial cell seeding density of MSCs has an effect on bone tissue formation in vitro [11]. In our study, we used an initial cell seeding density of 1 million cells/100 mg scaffold, which is in accordance with other studies showing that a higher cell density has no beneficial effects on the bone forming abilities [11,45].…”

Spatiotemporal proliferation of human stromal cells adjusts to nutrient availability and leads to stanniocalcin-1 expression in vitro and in vivo

“…We chose PEOTPBT because its biocompatibility has been extensively studied and the processing of PEOT/PBT biomaterials into a 2D system as a film and into a 3D system as a scaffold enabled us to control three physical parameters: cell seeding density, pore geometry and shear stress. The cell seeding density is important because it has an effect on the number of cell divisions [28] and differentiation potential [4] of MSCs in culture and their ability to regenerate tissue, such as bone [29]. Therefore, the seeding density was controlled through the surface area in both 2D and 3D.…”

Mesenchymal properties of SJL mice-stem cells and their efficacy as autologous therapy in a relapsing–remitting multiple sclerosis model

“…The biological characteristics with which cell populations must comply to be defined as MSCs are clear, regardless of the tissue of origin [ 10 , 11 ]. In recent years, several studies have described isolation protocols, culture, expansion, phenotypic and functional characterization of human and animal MSCs derived from different tissues [ 12 – 17 ]. However, although these cells are assumed to be similar, contradictory results have been obtained revealing interspecies/intraspecies variations regarding cell morphology, cell survival and senescence [ 18 ], cell proliferation, surface marker profile, growth kinetics [ 9 , 12 , 15 ], differentiation capacity [ 3 , 16 ], gene expression [ 13 ], genetic instability or even malignant transformation [ 14 ], among others.…”