Coculture with monocytes/macrophages modulates osteogenic differentiation of adipose-derived mesenchymal stromal cells on poly(lactic-co-glycolic) acid/polycaprolactone scaffolds

Abstract

The effects of immune cells, in particular macrophages, on the behaviour of mesenchymal stromal cells (MSCs) have recently gained much attention for MSCs-based tissue-engineered constructs. This study aimed to evaluate the effect of monocytes/macrophages on the osteogenic differentiation of adipose-derived mesenchymal stromal cells (ADMSCs) in three-dimensional (3D) cocultures. For this, we cocultured THP-1 monocytes, M1 macrophages, or M2 macrophages with ADMSCs on 3D poly(lactic-co-glycolic) acid (PLGA)/polycaprolactone (PCL) scaffolds using osteogenic medium for up to 42 days. We found that osteogenic differentiation of ADMSCs was inhibited by monocytes and both macrophage subtypes in 3D scaffolds. Furthermore, coculture of monocytes/macrophages with ADMSCs resulted in downregulated secretion of oncostatin M (OSM) and bone morphogenetic protein 2 (BMP-2) and inhibited expression of osteogenic markers alkaline phosphatase (ALP), bone sialoprotein (BSP), and runt-related transcription factor 2 (RUNX2). Compared with both macrophage subtypes, monocytes inhibited osteogenic differentiation of ADMSCs more significantly. These data suggest that the mutual interactions between monocytes/macrophages and ADMSCs negatively affect MSC osteogenic differentiation and thus possibly bone healing capacity, which highlights the importance of the micro-environment in influencing cell-based constructs to treat bone defects and the potential to improve their performance by resolving the inflammation ahead of treatment.

Keywords: 3D; adipose-derived mesenchymal stromal cells; coculture; macrophages; monocytes; osteogenic differentiation.

Figure 1

Schematic representation of the experimental design. ADMSCs: adipose‐derived mesenchymal stromal cells; ALP: alkaline phosphatase; TNF‐α: tumour necrosis factor alpha; BMP‐2: bone morphogenetic protein 2; BSP: bone sialoprotein; COL1: collagen type 1; DAPI: 4′,6‐diamidino‐2‐phenylindole; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; HE: haematoxylin and eosin; OSM: oncostatin M; PCL: polycaprolactone; PLGA: poly(lactic‐co‐glycolic) acid; RT‐PCR: reverse transcription polymerase chain reaction; RUNX2: runt‐related transcription factor 2; TGF‐β: transforming growth factor β [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Characterization of adipose‐derived mesenchymal stromal cells (ADMSCs), M1, and M2 macrophages. (a–d) The ratio of positive cells in ADMSCs was compared with the ratio in negative controls. Gates were set using the negative control. Red lines indicate the histogram for ADMSCs markers, and green lines indicate the histogram for negative controls. (e) M1 macrophages and M2 macrophages were stained with M1 marker CCR7 (red), M2marker CD36 (green), and 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue). (f) Quantification of relative fluorescence intensity of CCR7 and CD36 in M1 macrophages and M2 macrophages. (g) The concentration of tumour necrosis factor alpha (TNF‐α) and transforming growth factor β (TGF‐β) in M1 and M2 macrophage culture medium was measured by ELISA. Scale bar, 50 μm. “*” indicates significant difference between groups. *p < 0.05, **p < 0.01, ***p < 0.001 [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

(a) Cell seeding efficiency in each group. (b) DNA content in each group on Days 1, 3, 7, 14, and 28. (c) Gross alkaline phosphatase (ALP) activity in each group on Days 3, 7, 14, and 28. (d) Calcium content in each group on Days 14, 28, and 42. “*” indicates significant difference compared with the adipose‐derived mesenchymal stromal cell (ADMSC) group at the same time point. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 4

Cytokine secretion analysis by ELISA. (a) Corrected transforming growth factor β (TGF‐β) concentration in each group on Days 3, 7, 14, 28, and 42. (b) Corrected tumour necrosis factor alpha (TNF‐α) concentration in each group on Days 3, 7, 14, 28, and 42. (c) Corrected oncostatin M (OSM) concentration in each group on Days 3, 7, 14, 28, and 42. (d) Corrected bone morphogenetic protein 2 (BMP‐2) concentration in each group on Days 3, 7, 14, 28, and 42. “*” indicates significant difference compared with the adipose‐derived mesenchymal stromal cell (ADMSC) group at the same time point. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 5

Gene expression of osteogenic markers. (a) Gene expression of alkaline phosphatase (ALP) in each group on Days 3, 7, 14, and 28. (b) Gene expression of bone sialoprotein (BSP) in each group on Days 3, 7, 14, and 28. (c) Gene expression of collagen type 1 (COL1) in each group on Days 3, 7, 14, and 28. (d) Gene expression of osteocalcin (OCN) in each group on Days 3, 7, 14, and 28. (e) Gene expression of runt‐related transcription factor 2 (RUNX2) in each group on Days 3, 7, 14, and 28. “*” indicates significant difference compared with the adipose‐derived mesenchymal stromal cell (ADMSC) group at the same time point. *p < 0.05, **p < 0.01, ***p < 0.001. “#” indicates significant difference compared with Day 3 in the same group. #p < 0.05, ##p < 0.01, ###p < 0.001. “+” indicates significant difference compared with Day 7 in the same group. +p < 0.05, ++p < 0.01, +++p < 0.001. “$” indicates significant difference compared with Day 14 in the same group. $p < 0.05, $$p < 0.01, $$$p < 0.001