Substrate nanotexture and hypergravity through centrifugation enhance initial osteoblastogenesis

Abstract

Mimicking the structural nanomolecular extracellular matrix with synthetically designed nanosized materials is a relatively new approach, which can be applied in the field of bone tissue engineering. Likewise, bone tissue-engineered constructs can be aided in their development by the use of several types of mechanical stimuli. In this study, we wanted to combine nanotextured biomaterials and centrifugation in one multifactorial system. Mesenchymal stem cells were isolated from rat bone marrow, and cultured on a nanogrooved polystyrene substrate (200-nm-wide pitch with a depth of 50 nm). Constant centrifugation of 10 g was applied to cells up to 7 days. Results showed that on a nanogrooved substrate osteoblast-like cells align parallel to the groove direction. Centrifugation of 10 g also affected cell morphology on a smooth surface. Moreover, cell alignment was significantly reduced for cells grown on nanogrooved substrates, which were subsequently subjected to centrifugation. Independently, both stimuli increased the number of cells after 7 days of culture. However, when both stimuli were combined, an additive effect on cell number was observed, followed by an enhanced effect on osteocalcin mRNA expression and matrix mineralization. In conclusion, biomaterial surface modification as well as centrifugation are effective means to enhance bone cell behavior, moreover, readily available to many tissue engineers.

FIG. 1.

General setup. (a) Polystyrene (PS) dishes seeded with cells were placed in a custom-made PS holder in a 50-mL tube equipped with a silicone rubber stopper on the bottom. (b) The Large Diameter Centrifuge (LDC) can provide hypergravity of 10 g. The system comprises of large rotating arms, where a swing-out gondola is attached at the extremity, and a central gondola serves as a rotational control. Color images available online at www.liebertpub.com/tea

FIG. 2.

(a and b) AFM topographies of the nanogrooved PS substrates confirmed that the pattern of grooves and ridges were well reproduced in PS, with an average pitch of 207.3±1.5 nm, and a depth of 50.1±5.5 nm.

FIG. 3.

Scanning electron micrographs of osteoblast-like cells grown under various conditions for 1 day at 500× magnification. Top left: cells, grown on a smooth substrate at 1 g have few cellular extensions and top right: cells grown on a nanotextured substrate with extensions aligned parallel to the nanogrooves, insert in the corner is 2000×magnification of the nanogrooves beneath the cells. Bottom left: cells grown on a smooth substrate in hypergravity have more extensions and bottom right: cells grown on a nanotextured surface with branched, but also longer cellular extensions in multiple directions. White arrows indicate the direction of the nanogrooves and the letter c is abbreviation for cell.

FIG. 4.

Scanning electron microscopy images after 7 days of cell culture at 200× magnification. Note the onset of mineralization. Only the mineral particles on the nanotextured substrate in hypergravity were deposited in an aligned mode. Inserts in the corners show: 2000× magnification the nanogrooves and mineral particles. White arrows indicate the direction of the nanogrooves and the letter c is abbreviation for cell.

FIG. 5.

Cellular morphological characteristics. (a) Fluorescent microscopy graphs of actin staining (red) and nuclei staining (blue) of osteoblast-like cells grown for 48 h. Upper part of the graph: cells grown on a smooth substrate with random cell and actin orientation at 1 g and 10 g, respectively. Lower part of the graph: note that the parallel cell and actin orientation to the nanogrooves in normal gravity is slightly lost in hypergravity. White arrow indicates the direction of the nanogrooves (b). Median angle of ∼ 50° is a random cell orientation on the smooth substrates. Note that cells align in parallel direction to the nanogrooves (p<0.001) in normal gravity, and that it changes significantly in hypergravity increasing after 24 h (p<0.001) and 48 h (p<0.01). (c) Box-Whisker plot with cell elongation ratio measurements (height/width ratio) showed that cells cultured on the smooth substrate have rounded bodies. Cells grown on the nanogrooved substrate after 24 h had more elongated bodies and in hypergravity the elongation ratio of the cells dropped significantly after 24 h (p<0.01) and 48 h (p<0.05). (d) Box-Whisker plot showing the surface area of the cells. Overall cell area increased significantly over time on the smooth substrate in normal gravity and in hypergravity (p<0.001). Note that the median cell area on the nanotextured substrate is significantly smaller in all three time points in both groups when compared with the smooth substrates, respectively. Each box represents ∼200 cells (*p<0.01, **p<0.001).

FIG. 6.

(a) Cell proliferation graph by a total DNA content measurement (in μg/mL−1). The error bars represent the standard deviations. In normal conditions, at day 1, no significant difference in the total amount of DNA was observed. At day 7 in normal conditions, significant increase of cell number (p<0.05) on the nanotexture substrates was observed when compared to the smooth substrate. The same result was also observed in hypergravity, an additive effect was observed when cells were grown on the nanotextured substrate in hypergravity. (b) The Ca content was significantly higher only for cells grown on the nanotextured substrate under hypergravity conditions. (*p<0.05, **p<0.01, ***p<0.001) n=1. Color images available online at www.liebertpub.com/tea

FIG. 7.

Influence of nanotexture, hypergravity, and combination of both on gene expression evaluated after day 1 and day 7 of cell culture. Values were normalized to GAPDH and HPRT and relative to the smooth substrates (black bar or gray horizontal line). (a) Apoptosis-related genes BCL2, BAX, and p53 did not show any significant upregulation or downregulation neither at day 1, or day 7. (b) Osteoblastogenesis genes. At day 1, only OC was significantly upregulated on the nanotextured substrate in hypergravity. Same result was observed at day 7. In contrast, RUNX2 was significantly upregulated on the nanotextured substrate in normal conditions, but also on the smooth substrate in hypergravity. β-cat was only significantly up regulated only on the nanotextured substrate (*p<0.05, **p<0.01) n=3. Color images available online at www.liebertpub.com/tea