Dose effect of tumor necrosis factor-alpha on in vitro osteogenic differentiation of mesenchymal stem cells on biodegradable polymeric microfiber scaffolds

Abstract

This study presents a first step in the development of a bone tissue engineering strategy to trigger enhanced osteogenesis by modulating inflammation. This work focused on characterizing the effects of the concentration of a pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-alpha), on osteogenic differentiation of mesenchymal stem cells (MSCs) grown in a 3D culture system. MSC osteogenic differentiation is typically achieved in vitro through a combination of osteogenic supplements that include the anti-inflammatory corticosteroid dexamethasone. Although simple, the use of dexamethasone is not clinically realistic, and also hampers in vitro studies of the role of inflammatory mediators in wound healing. In this study, MSCs were pre-treated with dexamethasone to induce osteogenic differentiation, and then cultured in biodegradable electrospun poly(epsilon-caprolactone) (PCL) scaffolds, which supported continued MSC osteogenic differentiation in the absence of dexamethasone. Continuous delivery of 0.1 ng/mL of recombinant rat TNF-alpha suppressed osteogenic differentiation of rat MSCs over 16 days, which was likely the result of residual dexamethasone antagonizing TNF-alpha signaling. Continuous delivery of a higher dose, 5 ng/mL TNF-alpha, stimulated osteogenic differentiation for a few days, and 50 ng/mL TNF-alpha resulted in significant mineralized matrix deposition over the course of the study. These findings suggest that the pro-inflammatory cytokine TNF-alpha stimulates osteogenic differentiation of MSCs, an effect that can be blocked by the presence of anti-inflammatory agents like dexamethasone, with significant implications on the interplay between inflammation and tissue regeneration.

Figure 1

Electrospun microfibers generated for this study, imaged via SEM at varying magnifications, (a) 200×, (b) 600×, and (c) 2000×. A cross-section through the entire thickness of a representative scaffold is shown in (d), at 120× magnification. Scale bars shown for (a) and (b) represent 100 μm, 20 μm for (c), and 200 μm for (d).

Figure 2

Cellularity of MSC-seeded electrospun PCL scaffolds after 4, 8, and 16 days of culture. Three freeze/thaw/sonication cycles were used to extract dsDNA, which was then quantified with a fluorometric kit and translated to cells/scaffold. Experimental groups were cultured in dexamethasone-free osteogenic media supplemented with 0.1, 5, or 50 ng/mL TNF-α (abbreviated “TNF” in the figure). Three control groups were included: a positive control supplemented with dexamethasone, but no TNF-α (“0 ng/mL TNF +dex”); a negative control cultured without any added dexamethasone (“0 ng/mL TNF”); and an acellular control, where cell-free PCL scaffolds were cultured in dexamethasone-free media (“Acellular”). Each bar represents the mean ± standard deviation for n = 4 constructs. Error bars are included for all groups, though they are too small to resolve for “Acellular.” Statistical differences (p < 0.05) among groups at a single timepoint are indicated by “*” for a group that differs from all other groups, and “**” for a group that differs from only some groups. Changes over time are indicated by “#,” meaning that values for that group at each timepoint are significantly different (p < 0.05).

Figure 3

Alkaline phosphatase (ALP) activity of MSC-seeded electrospun PCL scaffolds after 4, 8, and 16 days of culture. Experimental groups were cultured in dexamethasone-free osteogenic media supplemented with 0.1, 5, or 50 ng/mL TNF-α (abbreviated “TNF” in the figure). Three control groups were included: a positive control supplemented with dexamethasone, but no TNF-α (“0 ng/mL TNF +dex”); a negative control cultured without any added dexamethasone (“0 ng/mL TNF”); and an acellular control, where cell-free PCL scaffolds were cultured in dexamethasone-free media (“Acellular”). Each bar represents the mean ± standard deviation for n = 4 constructs. Error bars are included for all groups, though they are too small to resolve in some cases. Statistical differences (p < 0.05) among groups at a single timepoint are indicated by “*” for a group that differs from all other groups, and “**” for a group that differs from only some groups. Changes over time are indicated by “#,” meaning that values for that group at each timepoint are significantly different (p < 0.05).

Figure 4

Calcium content of MSC-seeded electrospun PCL scaffolds after 4, 8, and 16 days of culture. Experimental groups were cultured in dexamethasone-free osteogenic media supplemented with 0.1, 5, or 50 ng/mL TNF-α (abbreviated “TNF” in the figure). Three control groups were included: a positive control supplemented with dexamethasone, but no TNF-α (“0 ng/mL TNF +dex”); a negative control cultured without any added dexamethasone (“0 ng/mL TNF”); and an acellular control, where cell-free PCL scaffolds were cultured in dexamethasone-free media (“Acellular”). Each bar represents the mean ± standard deviation for n = 4 constructs. Error bars are included for all groups, though they are too small to resolve in some cases. Statistical differences (p < 0.05) among groups at a single timepoint are indicated by “*” for a group that differs from all other groups, and “**” for groups that do not differ from each other, but differ from all other groups. Changes over time are indicated by “#,” meaning that values for that group at each timepoint are significantly different (p < 0.05).

Figure 5

Representative cross-sectional images (5 μm thick) of stained MSC-seeded electrospun PCL scaffolds after 4, 8, and 16 days of culture. Sections were stained with von Kossa and eosin. Each image shows only the top half of each cross-section, which is where cells (representative examples indicated by yellow arrows) and mineral deposits (representative examples marked with blue arrows) were located. Experimental groups were cultured in dexamethasone-free osteogenic media supplemented with 0.1, 5, or 50 ng/mL TNF-α (abbreviated “TNF” in the figure). Three control groups were included: a positive control supplemented with dexamethasone, but no TNF-α (“0 ng/mL TNF +dex”); a negative control cultured without any added dexamethasone (“0 ng/mL TNF”); and an acellular control, where cell-free PCL scaffolds were cultured in dexamethasone-free media (“Acellular”). Images were captured at 10× original magnification. Scale bar in lower right corner represents 200 μm and applies to all images.

Figure 5

Representative cross-sectional images (5 μm thick) of stained MSC-seeded electrospun PCL scaffolds after 4, 8, and 16 days of culture. Sections were stained with von Kossa and eosin. Each image shows only the top half of each cross-section, which is where cells (representative examples indicated by yellow arrows) and mineral deposits (representative examples marked with blue arrows) were located. Experimental groups were cultured in dexamethasone-free osteogenic media supplemented with 0.1, 5, or 50 ng/mL TNF-α (abbreviated “TNF” in the figure). Three control groups were included: a positive control supplemented with dexamethasone, but no TNF-α (“0 ng/mL TNF +dex”); a negative control cultured without any added dexamethasone (“0 ng/mL TNF”); and an acellular control, where cell-free PCL scaffolds were cultured in dexamethasone-free media (“Acellular”). Images were captured at 10× original magnification. Scale bar in lower right corner represents 200 μm and applies to all images.