Inhibition of ERK promotes collagen gel compaction and fibrillogenesis to amplify the osteogenesis of human mesenchymal stem cells in three-dimensional collagen I culture

Abstract

Tissue morphogenesis remains one of the least understood problems in cell and developmental biology. There is a disconnect between the mechanisms that apply to two-dimensional (2D) cultures and those seen in vivo. Three-dimensional (3D) culture presents a complex stimulus triggering cellular responses that are only partially understood. We compared 2D and 3D cultures of human mesenchymal stem cells in the presence of mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, to determine the role of extracellular signal-related kinase (ERK) in collagen-induced differentiation. 3D collagen I culture enhanced and accelerated the osteogenic differentiation of human mesenchymal stem cells (hMSC). Contrary to 2D results, the addition of PD98059 induced a significant amplification of osteogenic gene expression and matrix mineralization in 3D cultures. The inhibition of ERK altered cell-mediated compaction, proliferation, and resulted in the development of distinct tissue microstructure. Therefore, we suggest that the ability to reorganize collagen in 3D is an important step in ERK-mediated osteogenic differentiation. This work aims to propose a correlation between osteogenic differentiation and hMSC-directed collagen I remodeling. We present a potential mechanistic link (ERK) through which the three dimensionality of an engineered tissue acts to differentially induce and maintain cellular phenotype during tissue development.

FIG. 1.

3D culture of human mesenchymal stem cells (hMSCs) in collagen I hydrogels results in enhanced osteogenic differentiation. hMSC cultured in 3D collagen I hydrogels were assayed for the expression of five markers of osteogenic differentiation at days 1, 3, 7, and 14. (A) Bone sialoprotein (BSP), (B) osteocalcin (BGLAP), (C) runt-related transcription factor 2 (RUNX2), (D) osterix (OSX), and (E) collagen I (COL I). Samples are normalized to GAPDH and shown as a fold increase in gene expression from hMSC plated on 2D TCP at day 1. ^*Statistical significance p < 0.05, n = 3 relative to 2D hMSC plated on TCP.

FIG. 2.

3D collagen I induces a range of human mesenchymal stem cell (hMSC) gene response. hMSC cultured in 3D collagen I hydrogels were assayed for the expression of markers of the chondrogenic, myogenic, and adipogenic lineages to assess nonosteogenic differentiation in response to the 3D collagen I stimulus. Chondrogenic: (A) collagen II (COL II) and (B) aggrecan (ACAN); myogenic: (C) myogenin (MYOG) and (D) dystrophin (DMD); and adipogenic: (E) fatty acid binding protein 4 (FABP4) and (F) peroxisome proliferator-activated receptor γ (PPARG). Samples are normalized to GAPDH and shown as a fold increase in gene expression from hMSC plated on 2D TCP at day 1. ^*Statistical significance p ≤ 0.05, n = 3 relative to 2D hMSC plated on TCP.

FIG. 3.

The addition of the MEK inhibitor PD98059 decreases ERK activity. ERK activity was measured using an ELISA assay kit and quantified as pg/μL of phosphorylated ERK (pERK). Cell lysate was harvested in RIPA supplemented with protease inhibitors. Lysate was loaded into the assay and normalized to cell number. pERK was quantified through detection of substrate conversion at an OD of 450 nm. ^*Statistical significance p ≤ 0.05, n = 3.

FIG. 4.

(A) and B) PD98059 alters the compaction of collagen gels. hMSC were embedded and cultured within collagen I hydrogels and observed over time. The area of each gel was calculated by measurement of the x and y diameter at days 1, 3, and 7. Area was calculated using the following equation: A = πr². ^*Statistical significance p ≤ 0.05, n ≥ 12.

FIG. 5.

(A) PD98059 does not affect 3D human mesenchymal stem cell proliferation. Cellular number was assessed in both 2D and 3D conditions with and without the drug, PD98059, at days 0, 7, and 21. (A) 2D cell numbers at each time point were assessed by counting adherent cells. (B) 3D cell number was assessed using the DNA Hoescht assay. ^*Statistical significance p ≤ 0.05, n = 3. Error bars in (A) are too tight to be resolved.

FIG. 6.

(A) PD98059 amplifies the osteogenic differentiation of hMSC in 3D collagen I culture. hMSC embedded in 3D collagen I were cultured for 7 days and then assayed for (A) osteogenic (BSP, BGLAP, RUNX2, OSX, COL I), (B) chondrogenic (COL II, ACAN), and myogenic (MYOG, DMD) lineage markers. Evaluated gene expression was normalized to GAPDH and expressed here as fold gene expression over 2D TCP controls. ^*Statistical significance p ≤ 0.05, n ≥ 3.

FIG. 7.

(A) PD98059 increases the deposition of mineral by human mesenchymal stem cells (hMSCs) in 3D collagen I culture. hMSCs were again embedded in 3D collagen I and cultured for 21 days to assess matrix mineralization. Frozen sections were mounted and stained using alizarin red S (ARS) stain for calcium deposition. Scale bars represent 20 μm and images were taken at 40× magnification.

FIG. 8.

(A) PD98059 promotes the rearrangement of the tissue microstructure. Human mesenchymal stem cells were embedded in 3D collagen I and cultured for 21 days to assess tissue morphology. Samples were dehydrated, sectioned using a cryostat, and mounted for imaging. (A–C) Sections of untreated, DMSO, and PD98059 samples were stained with H&E to distinguish cellular microarchitecture within the developing “tissue.” (D–F) Additional mounted sections were imaged using SHG confocal microscopy to evaluate the degree of collagen I network organization. Samples were all excited at a wavelength of 820 nm and signal was collected at 410 nm equally for each sample. Images are representative of a larger set, n ≥ 3 and were taken at a 40× magnification. Scale bars represent 20 μm.