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. 2015 Feb;41:79-88.
doi: 10.1016/j.biomaterials.2014.11.020. Epub 2014 Dec 5.

Immunomodulation by Mesenchymal Stem Cells Combats the Foreign Body Response to Cell-Laden Synthetic Hydrogels

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Free PMC article

Immunomodulation by Mesenchymal Stem Cells Combats the Foreign Body Response to Cell-Laden Synthetic Hydrogels

Mark D Swartzlander et al. Biomaterials. .
Free PMC article

Abstract

The implantation of non-biological materials, including scaffolds for tissue engineering, ubiquitously leads to a foreign body response (FBR). We recently reported that this response negatively impacts fibroblasts encapsulated within a synthetic hydrogel and in turn leads to a more severe FBR, suggesting a cross-talk between encapsulated cells and inflammatory cells. Given the promise of mesenchymal stem cells (MSCs) in tissue engineering and recent evidence of their immunomodulatory properties, we hypothesized that MSCs encapsulated within poly(ethylene glycol) (PEG) hydrogels will attenuate the FBR. In vitro, murine MSCs encapsulated within PEG hydrogels attenuated classically activated primary murine macrophages by reducing gene expression and protein secretion of pro-inflammatory cytokines, most notably tumor necrosis factor-α. Using a COX2 inhibitor, prostaglandin E2 (PGE2) was identified as a mediator of MSC immunomodulation of macrophages. In vivo, hydrogels laden with MSCs, osteogenically differentiating MSCs, or no cells were implanted subcutaneously into C57BL/6 mice for 28 days to assess the impact of MSCs on the fibrotic response of the FBR. The presence of encapsulated MSCs reduced fibrous capsule thickness compared to acellular hydrogels, but this effect diminished with osteogenic differentiation. The use of MSCs prior to differentiation in tissue engineering may therefore serve as a dynamic approach, through continuous cross-talk between MSCs and the inflammatory cells, to modulate macrophage activation and attenuate the FBR to implanted synthetic scaffolds thus improving the long-term tissue engineering outcome.

Keywords: Foreign body response; Hydrogel; Immunomodulation; Macrophage; Mesenchymal stem cell; Poly(ethylene glycol).

Figures

Figure. 1
Figure. 1
Effect of MSCs on macrophage (Mϕ) activation. a) Experimental design for in vitro studies. b) Relative gene expression and cytokine secretion of pro-inflammatory cytokines, TNF-α and IL-6, in macrophage and MSCs monocultures and co-culture in the absence and presence of the inflammatory stimulant, LPS. c) Relative gene expression of arginase in macrophage and MSC monocultures and co-culture in the absence and presence of LPS. Gene expression is relative to the housekeeping gene, L32. Data are presented as mean with standard deviation as error bars (n=4). * p<0.05, ** p<0.01, and *** p<0.001.
Figure. 2
Figure. 2
Role of PGE2 in MSC immunomodulation of LPS-activated macrophages (Mϕ). a) Secretion of PGE2 from macrophages and MSC monocultures and co-culture in the absence and presence of LPS. b) Macrophage cytokine secretion of pro-inflammatory cytokines, TNF-α and IL-6, with increasing concentrations of PGE2. c) MSC secretion of PGE2 in the presence of increasing amounts of a cyclooxygenase 2 (COX2) inhibitor. d) MSC secretion of pro-inflammatory cytokines, TNF-α and IL-6, in the absence (gray) and presence (green) of a COX2 inhibitor. e) Macrophage secretion of pro-inflammatory cytokines, TNF-α and IL-6, with increasing amounts of normal MSC conditioned medium (gray) and MSC conditioned medium with a COX2 inhibitor (green). Data are presented as mean with standard deviation as error bars (n=4).). * p<0.05, ** p<0.01, and *** p<0.001.
Figure. 3
Figure. 3
Proposed mechanism of MSC-driven attenuation of LPS-activated macrophages (Mϕ). a) Proposed mechanism in macrophages where red lines indicate signaling by LPS, an inflammatory stimulant and black lines indicating signaling by PGE2 signaling. b) Immunocytochemistry analysis of macrophages seeded onto acellular hydrogels. Macrophages were stained for β-catenin (green) in the presence of LPS alone, or LPS with PGE2 or a GSK-3β inhibitor. Nuclei were stained with DAPI (blue). Scale bar is 20 µm. c) c-myc gene expression in macrophages on PEG hydrogels in the absence and presence of LPS and with and without encapsulated MSCs at 4 hours. Gene expression is relative to the housekeeping gene, L32. Data are presented as mean with standard deviation as error bars (n=4). * p<0.05, ** p<0.01, and *** p<0.001.
Figure. 4
Figure. 4
Effect of MSC differentiation on macrophage (Mϕ) activation. a) Confirmation of osteogenic differentiation of murine MSCs by alkaline phosphatase (ALP) activity over the 21 days differentiation period normalized to total DNA content and by Alizarin Red S staining for mineral deposits. b) Experimental design for the differentiation and encapsulation of MSCs. c) Relative gene expression in macrophages cultured on PEG hydrogels laden with MSCs and osteogenically differentiating MSCs in the presence of LPS for pro-inflammatory cytokines, TNF-α and IL-6, and d) arginase. Gene expression is relative to the housekeeping gene, L32. Data are presented as mean with standard deviation as error bars (n=4). * p<0.05, ** p<0.01, and *** p<0.001. Scale bar equals 100µm.
Figure. 5
Figure. 5
In vivo response to PEG hydrogels laden with MSCs and osteogenically differentiating MSCs. Hydrogels were implanted subcutaneously for 28 days in immunocompetent C57bl/6 mice. Masson’s Trichrome staining and H&E staining for PEG hydrogels containing: MSCs, 4 day differentiated MSCs, 10 day differentiated MSCs, 21 day differentiated MSCs, and no cells (referred to as PEG). Arrows in Masson’s Trichrome stained sections indicate the fibrous capsule, while arrows in the H&E stained sections indicate the layer of inflammatory cells at the material / host interface. Fibrous capsule and inflammatory cell layers thicknesses are given as the number of cells layers present. Data are presented as mean with standard deviation as error bars (n=4). * p<0.05, ** p<0.01, and *** p<0.001. Scale bar is 100 µm.

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