The osteogenic differentiation of human bone marrow MSCs on HUVEC-derived ECM and β-TCP scaffold

Abstract

Extracellular matrix (ECM) serves a key role in cell migration, attachment, and cell development. Here we report that ECM derived from human umbilical vein endothelial cells (HUVEC) promoted osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSC). We first produced an HUVEC-derived ECM on a three-dimensional (3D) beta-tricalcium phosphate (β-TCP) scaffold by HUVEC seeding, incubation, and decellularization. The HUVEC-derived ECM was then characterized by SEM, FTIR, XPS, and immunofluorescence staining. The effect of HUVEC-derived ECM-containing β-TCP scaffold on hMSC osteogenic differentiation was subsequently examined. SEM images indicate a dense matrix layer deposited on the surface of struts and pore walls. FTIR and XPS measurements show the presence of new functional groups (amide and hydroxyl groups) and elements (C and N) in the ECM/β-TCP scaffold when compared to the β-TCP scaffold alone. Immunofluorescence images indicate that high levels of fibronectin and collagen IV and low level of laminin were present on the scaffold. ECM-containing β-TCP scaffolds significantly increased alkaline phosphatase (ALP) specific activity and up-regulated expression of osteogenesis-related genes such as runx2, alkaline phosphatase, osteopontin and osteocalcin in hMSC, compared to β-TCP scaffolds alone. This increased effect was due to the activation of MAPK/ERK signaling pathway since disruption of this pathway using an ERK inhibitor PD98059 results in down-regulation of these osteogenic genes. Cell-derived ECM-containing calcium phosphate scaffolds is a promising osteogenic-promoting bone void filler in bone tissue regeneration.

Fig. 1

(A) The schematic of HUVEC-derived ECM production. Left: lines depict ECM, and cells are on the ECM basement; right: ECM after the extraction procedure. (B) Light microscopy images of HUVEC cultured on a tissue culture plate for 14 days after confluence before (left) and after (right) decellularization (right). Location “+” marked identical fields. (C) Fluorescent image of HUVEC cultured on the β-TCP scaffold for 14 days before (left) and after (right) decellularization. (Magnification: 4×).

Fig. 2

SEM images of β-TCP scaffold before seeding the HUVEC (left) and of the ECM layer on the β-TCP scaffold after decellularization (right).

Fig. 3

Immunofluorescent staining images of collagen type IV, fibronectin, and laminin before and after decelluarization. Red: Alexa Fluor® 594 goat anti-mouse antibody; Blue: DAPI.

Fig. 4

ATR-FTIR transmission spectra of the β-TCP, ECM from tissue culture plate, and ECM/β-TCP. The characteristic peaks of amide and hydroxyl groups are shown on the ECM and ECM/β-TCP spectrum, respectively.

Fig. 5

XPS survey scan spectra (a) and XPS C1s core level spectra (b) for β-TCP, ECM from tissue culture plate, and ECM/β-TCP.

Fig. 6

The amount of dsDNA content synthesized by hMSC grown over time on β-TCP and ECM/β-TCP scaffolds (a). ALP activity of the hMSC on β-TCP and ECM/β-TCP scaffolds (b) (n=3). An * and ** are marked to show significant differences between groups (*p<0.05, **p<0.01).

Fig. 7

Related osteogenic gene expression levels in hMSC cultured on β-TCP and ECM/β-TCP scaffolds. Runx2, alp, opn and oc gene expression levels were assessed by real time-PCR at day 7 and 14. GAPDH expression was also determined as an internal control. Significant difference between the two groups are shown as * and ** (*p<0.05, **p<0.01).

Fig. 8

Confocal microscope images of osteocalcin in hMSC cultured on β-TCP and ECM/β-TCP scaffolds for 14 and 21 days. Red: Alexa Fluor® 594 goat anti-mouse; Blue: DAPI.

Fig. 9

ALP activity of the hMSC on β-TCP and ECM/β-TCP scaffolds before and after the addition of inhibitor PD98059.

Fig. 10

Related osteogenic gene expression levels in hMSC cultured on ECM/β-TCP scaffolds with and without the presence of inhibitor PD98059. Runx2, alp, opn and oc gene expression levels were inhibited by the addition of PD98059. Significant difference between the two groups are shown as * and ** (*p<0.05, **p<0.01).

Fig. 11

Protein expression levels of phosphorylated ERK1/2 and total ERK1/2 were evaluated by Western blotting. Blocking MAPK/ERK signaling inhibits the osteogenic differentiation of hMSC on ECM/β-TCP scaffolds. Cell lysates were generated after 24 hours. The concentration of total protein was assayed by BCA assay.