Differentiation Capacity of Monocyte-Derived Multipotential Cells on Nanocomposite Poly(e-caprolactone)-Based Thin Films

Abstract

Background: Μonocyte-derived multipotential cells (MOMCs) include progenitors capable of differentiation into multiple cell lineages and thus represent an ideal autologous transplantable cell source for regenerative medicine. In this study, we cultured MOMCs, generated from mononuclear cells of peripheral blood, on the surface of nanocomposite thin films.

Methods: For this purpose, nanocomposite Poly(e-caprolactone) (PCL)-based thin films containing either 2.5 wt% silica nanotubes (SiO₂ntbs) or strontium hydroxyapatite nanorods (SrHAnrds), were prepared using the spin-coating method. The induced differentiation capacity of MOMCs, towards bone and endothelium, was estimated using flow cytometry, real-time polymerase chain reaction, scanning electron microscopy and fluorescence microscopy after cells' genetic modification using the Sleeping Beauty Transposon System aiming their observation onto the scaffolds. Moreover, Wharton's Jelly Mesenchymal Stromal Cells were cultivated as a control cell line, while Human Umbilical Vein Endothelial Cells were used to strengthen and accelerate the differentiation procedure in semi-permeable culture systems. Finally, the cytotoxicity of the studied materials was checked with MTT assay.

Results: The highest differentiation capacity of MOMCs was observed on PCL/SiO₂ntbs 2.5 wt% nanocomposite film, as they progressively lost their native markers and gained endothelial lineage, in both protein and transcriptional level. In addition, the presence of SrHAnrds in the PCL matrix triggered processes related to osteoblast bone formation.

Conclusion: To conclude, the differentiation of MOMCs was selectively guided by incorporating SiO₂ntbs or SrHAnrds into a polymeric matrix, for the first time.

Keywords: Monocyte-derived multipotential cells; Poly(ε-caprolactone); Silica nanotubes; Strontium hydroxyapatite nanorods.

Fig. 1

A Morphology of genetically modified normal WJSCs (a, b) and HUVEC (c, d) in cell culture. B Immunophenotypic characterization of WJSCs (a, b) and HUVEC (c) via flow cytometry

Fig. 2

A Comparative MOMCs culture in the presence of poly-l-lysine (upper) and fibronectin (below). B Immunophenotypic characterization of MOMCs after culture in the presence of poly-l-lysine and medium supplemented with SDF-1a

Fig. 3

A Testing of the NPs-mediated cytotoxicity in WJSCs cultures with MTT assay (a) 24 h and (b) 72 h after their addition in growth medium. B FTIR spectra of (a) PCL/SiO₂ntbs 2.5 wt% and (b), PCL/SrHAnrds 2.5 wt% in comparison to the spectra of their pure components

Fig. 4

Observation of morphological differentiation of A WJSCs and B MOMCs upon endothelial differentiation on different NP category PCL films

Fig. 5

A Immunophenotypic evaluation of the WJSCs (left) and MOMCs (right) differentiation towards endothelial cells. B Flow cytometry images of the CD45/CD144 marker expression before and after the completion of the WJSCs and MOMCs differentiation towards endothelium upon PCL/SiO₂ntbs films

Fig. 6

Real-time PCR for the evaluation of MOMCs and WJSCs differentiation in transcriptional level

Fig. 7

A, B SEM depiction of the WJSCs and MOMCs before and after their differentiation upon PCL films

Fig. 8

Evaluation of the differentiation capacity towards bone cells with the CPC method for MOMCS as well as WJSCs