Two stages in three-dimensional in vitro growth of tissue generated by osteoblastlike cells

Abstract

Bone regeneration is controlled by a variety of biochemical, biomechanical, cellular, and hormonal mechanisms. In particular, physical properties of the substrate such as stiffness and architecture highly influence the proliferation and differentiation of cells. The aim of this work is to understand the influence of scaffold stiffness and cell seeding densities on the formation of tissue by osteoblast cells within polyether urethane scaffolds containing pores of different sizes. MC3T3-E1 preosteoblast cells were seeded on the scaffold, and the amount of tissue formed within the pores was analyzed for culture times up to 49 days by phase contrast microscopy. The authors show that the kinetics of three-dimensional tissue growth in these scaffolds follows two stages and can be described by a universal growth law. The first stage is dominated by cell-material interactions with cell adherence and differentiation being strongly dependent on the polymer material. After a delay time of a few weeks, cells begin to grow within their own matrix, the delay being strongly dependent on substrate stiffness and seeding protocols. In this later stage of growth, three-dimensional tissue amplification is controlled rather by the pore geometry than the scaffold material properties. This emphasizes how geometric constraints may guide tissue formation in vitro and shows that optimizing scaffold architectures may improve tissue formation independent of the scaffold material used.

Availability of extracellular matrix biopolymers and differentiation state of human mesenchymal stem cells determine tissue-like growth in vitro

“…In line with our findings of correlating ECM amount and 3D tissue growth, an osteogenic cell line MCT3T-E1 previously used in similar experiments [35,36,42,52] was observed to secrete a very high amount of collagen (compared to MSC, data not shown) and subsequently rapidly created a stable 3D tissue-like structures inside various shaped macroscopic channels [39]. Nevertheless, both cell types, MC3T3-E1 and osteogenic differentiated MSC, showed an initial linear growth phase which turned into a plateau phase of growth after about 14 days.…”

“…Channel-containing culture carriers were previously used to investigate the influence of substrate geometry on bone like tissue growth [35]. After an initial migration, proliferation and adhesion phase till full coverage of the channel surfaces with cells, tissue growth kinetics in such systems was found independent of substrate characteristics, such as stiffness, most likely because further cell proliferation and differentiation occurs inside the cells own matrix [39]. Tissue growth was also found to be promoted by certain channel geometries [36].…”

The influence of curvature on three-dimensional mineralized matrix formation under static and perfused conditions: an in vitro bioreactor model

“…The ingrowth of mineralized tissue into the channel void, as represented by coverage, was directly dependent on curvature. As hypothesized, mineralized tissue formation was increased with increasing curvature, as reported for non-mineralized ECM previously [6,14,15,19]. A limitation of this study is the lack of a cell-free control group.…”

“…It was shown that there exists an intimate correlation between geometrical changes and cellular behaviour in vivo [3]. Different experimental studies have shown that curvature influences tissue growth under static and perfused conditions in vitro [6,[13][14][15]19]. Nevertheless, these studies focused on non-mineralized ECM formation only and evaluations of tissue growth were performed in limited three-dimensional volumes.…”

“…Despite the fact that three-dimensional scaffolds have been used to investigate the influence of curvature on tissue growth, analyses were performed using confocal microscopy limited to a penetration depth of about 500 mm [6,7,14,15,17,19]. To evaluate tissue growth along the whole height of the channels, it would have been necessary to cut the samples into thinner slices by destroying the samples, making them unavailable for longitudinal monitoring of tissue growth.…”