The in vitro effects of macrophages on the osteogenic capabilities of MC3T3-E1 cells encapsulated in a biomimetic poly(ethylene glycol) hydrogel

Abstract

Poly(ethylene glycol) PEG-based hydrogels are promising for cell encapsulation and tissue engineering, but are known to elicit a foreign body response (FBR) in vivo. The goal of this study was to investigate the impact of the FBR, and specifically the presence of inflammatory macrophages, on encapsulated cells and their ability to synthesize new extracellular matrix. This study employed an in vitro co-culture system with murine macrophages and MC3T3-E1 pre-osteoblasts encapsulated in a bone-mimetic hydrogel, which were cultured in transwell inserts, and exposed to an inflammatory stimulant, lipopolysaccharide (LPS). The co-culture was compared to mono-cultures of the cell-laden hydrogels alone and with LPS over 28 days. Two macrophage cell sources, RAW 264.7 and primary derived, were investigated. The presence of LPS-stimulated primary macrophages led to significant changes in the cell-laden hydrogel by a 5.3-fold increase in percent apoptotic osteoblasts at day 28, 4.2-fold decrease in alkaline phosphatase activity at day 10, and 7-fold decrease in collagen deposition. The presence of LPS-stimulated RAW macrophages led to significant changes in the cell-laden hydrogel by 5-fold decrease in alkaline phosphatase activity at day 10 and 4-fold decrease in collagen deposition. Mineralization, as measured by von Kossa stain or quantified by calcium content, was not sensitive to macrophages or LPS. Elevated interleukin-6 and tumor necrosis factor-α secretion were detected in mono-cultures with LPS and co-cultures. Overall, primary macrophages had a more severe inhibitory effect on osteoblast differentiation than the macrophage cell line, with greater apoptosis and collagen I reduction. In summary, this study highlights the detrimental effects of macrophages on encapsulated cells for bone tissue engineering.

Statement of significance: Poly(ethylene glycol) (PEG)-based hydrogels are promising for cell encapsulation and tissue engineering, but are known to elicit a foreign body response (FBR) in vivo. The impact of the FBR on encapsulated cells and their ability to synthesize tissue has not been well studied. This study utilizes thiol-ene click chemistry to create a biomimetic, enzymatically degradable hydrogel system with which to encapsulate MC3T3-E1 pre-osteoblasts. The osteogenic capabilities and differentiation of these cellswerestudied in co-culture with macrophages, known drivers of the FBR.This study demonstrates that macrophages reduce osteogenic capabilities of encapsulated cellsin vitroand suggestthat the FBR should be considered for in vivo tissue engineering.

Keywords: Co-culture; Host response; MC3T3-E1 pre-osteoblast; Macrophage; Osteogenesis; Poly(ethylene glycol) hydrogel.

Figure 1 –

Experimental setup. A) Schematic of the bone-mimetic hydrogel formation. B) Schematic of the experimental co-culture setup in this study, wherein macrophages were refreshed weekly and lipopolysaccharide (LPS) in the media was refreshed every 48 hours.

Figure 2 –

Characterization of the acellular hydrogel. A) Raman spectroscopy of hydrogels with 0 and 1% hydroxyapatite (HA), showing the indicative hydroxyapatite peak at 960 cm⁻¹. B) Compressive modulus and C) volumetric equilibrium swelling ratio of hydrogels with 0 and 1% hydroxyapatite hydrogels. Data are shown as mean (n=3) with standard deviation as error bars. D) Histogram of hydroxyapatite nanoparticle diameter, determined by FESEM.

Figure 3 –

The effects of macrophages on MC3T3-E1 cell apoptosis. A) Representative confocal microscopy images of hydrogels immediately after encapsulation and after 10 and 28 days of culture. Cells were stained for apoptosis (green) and counterstained with DAPI for cell nuclei (blue). Scale bar = 50 μm. B) Semi-quantification of the percent of apoptotic cells, normalized to number of nuclei in hydrogels after 0, 10, or 28 days of culture. C) DNA content per construct over time. Data are shown as mean (n=3–4) with standard deviation as error bars. “#” denotes statistical significance (p < 0.05) over day 0, “&” denotes statistical significance (p < 0.05) over day 10.

Figure 4 –

The effects of macrophages on MC3T3-E1 cell morphology. Representative confocal microscopy images of hydrogels immediately after encapsulation and after 10 days of culture. Cells were stained using calcein AM to visualize morphology. Red box indicates region of interest, white box shows ROI at 2x magnification. Scale bar = 100 μm.

Figure 5 –

The effect of macrophages on collagen I deposition by MC3T3-E1 cells. A) Representative confocal microscopy images of hydrogels after 0, 10, and 28 days of culture. Sections were stained for anti-collagen I (red) and counterstained with DAPI for cell nuclei (blue). Scale bar = 50 μm. B) Semi-quantification of the fluorescence (arbitrary units) normalized to the number of nuclei per image. Data are shown as mean (n=3–4) with standard deviation as error bars. P-values displayed are for pairwise comparisons between the unstimulated mono-culture and each treatment condition at each time point. “#” denotes statistical significance (p < 0.05) as compared to day 0, “&” denotes statistical significance (p < 0.05) as compared to corresponding condition at day 10.

Figure 6 –

The effects of macrophages on ALP activity and mineralization in MC3T3-E1-laden PEG hydrogels. A) ALP activity after 0, 10, and 28 days of culture. B) Total calcium content in hydrogels after 0, 10, and 28 days of culture. Blue dashed line indicates the mean calcium level at day 0, which provides a baseline for comparison. Data for A and B shown as mean (n = 3) with standard deviation as error bars. “#” denotes statistical significance (p < 0.05) as compared to day 0. C) Representative microscopy images of hydrogels fixed after 0, 10, and 28 days of culture and stained for von Kossa mineralization (black) and counterstained with nuclear fast red for nuclei (pink). Scale bar = 100 μm.

Figure 7 –

Cytokine secretion from the in vitro co-culture model. A) Interleukin-6 (IL-6) and B) tumor necrosis factor alpha (TNF-a) secretion in the media on day 10 and 28 as assessed by enzyme-linked immunosorbent assay (ELISA). Data are shown as mean (n=3–4) with standard deviation as error bars. Double dagger denotes undetectable levels.