Evaluating Oxygen Tensions Related to Bone Marrow and Matrix for MSC Differentiation in 2D and 3D Biomimetic Lamellar Scaffolds

Abstract

The physiological O₂ microenvironment of mesenchymal stem cells (MSCs) and osteoblasts and the dimensionality of a substrate are known to be important in regulating cell phenotype and function. By providing the physiologically normoxic environments of bone marrow (5%) and matrix (12%), we assessed their potential to maintain stemness, induce osteogenic differentiation, and enhance the material properties in the micropatterned collagen/silk fibroin scaffolds that were produced in 2D or 3D. Expression of osterix (OSX) and vascular endothelial growth factor A (VEGFA) was significantly enhanced in the 3D scaffold in all oxygen environments. At 21% O₂, OSX and VEGFA expressions in the 3D scaffold were respectively 13,200 and 270 times higher than those of the 2D scaffold. Markers for assessing stemness were significantly more pronounced on tissue culture polystyrene and 2D scaffold incubated at 5% O₂. At 21% O₂, we measured significant increases in ultimate tensile strength (p < 0.0001) and Young's modulus (p = 0.003) of the 3D scaffold compared to the 2D scaffold, whilst 5% O₂ hindered the positive effect of cell seeding on tensile strength. In conclusion, we demonstrated that the 3D culture of MSCs in collagen/silk fibroin scaffolds provided biomimetic cues for bone progenitor cells toward differentiation and enhanced the tensile mechanical properties.

Keywords: 2D vs. 3D; bone tissue engineering; mesenchymal stem cell; osteogenesis; oxygen tension.

Figure 1

For the physical characterization of the 2D and 3D collagen/silk fibroin scaffolds: stereomicroscope images were taken in the wet state for the (a) 2D (top view) and (b) 3D (side view) scaffolds. (c) Fluorescence microscopy and confocal laser scanning microscopy (CLSM) images to view the cross-section of the 3D scaffold after fluorescein labeling. SEM imaging of (d) 2D (scale bar: 20 μm) and (e) 3D scaffolds (scale bar: 200 μm). (f) SEM image of 3D scaffold’s cross-section (scale bar: 20 μm). The markings on the image indicated the micropattern dimensions.

Figure 2

Partial pressure of the O₂ (pO₂) inside the mesenchymal stem cell (MSC)-seeded 3D scaffold was determined at 5% and 21% O₂ tensions. An O₂ probe was inserted between the layers and into the core. After MSC seeding, O₂ monitoring was started and recorded until stationary pO₂ readings.

Figure 3

SEM micrographs at day 3 were taken to examine the topography conservation and MSC morphologies on the 2D and the 3D scaffold surfaces (scale bar: 10 μm).

Figure 4

Markers of osteogenic differentiation, stemness, and angiogenic factor were evaluated after 3 days of incubation at 5%, 12%, and 21% O₂ tensions. Transcript levels on TCPS, 2D scaffold, and in 3D scaffold were determined with real-time reverse-transcription polymerase chain reaction (real-time RT-PCR). Fine red lines indicate the normalized gene expression level, which is designated as 1. Osteogenic differentiation was studied with (a) runt-related transcription factor 2 (RUNX2) and (b) osterix (OSX). Stemness was examined with (c) bone marrow stromal cell antigen 1 (BST1) and (d) cluster of differentiation 90 (CD90). (e) Angiogenic activity was studied with vascular endothelial growth factor A (VEGFA).

Figure 5

Tensile properties of the 2D and the 3D scaffolds were determined after 35 days of incubation at 5% and 21% O₂ tensions. Unseeded and MSC-seeded samples were tested in the direction that was parallel to the microchannel (MC) axis. (a) Ultimate tensile strength (UTS) and (b) Young’s modulus (E) were presented as graphs. The average ± standard deviation values of (c) UTS and (d) E were given in tables. (e) Increase in UTS and (f) increase in E were also calculated.