The Role of Substrate Topography and Stiffness on MSC Cells Functions: Key Material Properties for Biomimetic Bone Tissue Engineering

Abstract

The hypothesis of the present research is that by altering the substrate topography and/or stiffness to make it biomimetic, we can modulate cells behavior. Substrates with similar surface chemistry and varying stiffnesses and topographies were prepared. Bulk PCL and CNTs-reinforced PCL composites were manufactured by solvent casting method and electrospinning and further processed to obtain tunable moduli of elasticity in the range of few MPa. To ensure the same chemical profile for the substrates, a protein coating was added. Substrate topography and properties were investigated. Further on, the feedback of Wharton's Jelly Umbilical Cord Mesenchymal Stem Cells to substrates characteristics was investigated. Solvent casting scaffolds displayed superior mechanical properties compared to the corresponding electrospun films. However, the biomimetic fibrous texture of the electrospun substrates induced improved feedback of the cells with respect to their viability and proliferation. Cells' adhesion and differentiation was remarkably pronounced on solvent casting substrates compared to the electrospun substrates. Soft substates improved cells multiplication and migration, while stiff substrates induced differentiation into bone cells. Aspects related to the key factors and the ideal properties of substrates and microenvironments were clarified, aiming towards the deep understanding of the required optimum biomimetic features of biomaterials.

Keywords: biocompatibility; bone tissue regeneration; mesenchymal stem cells; polymeric biomaterials; substrate stiffness.

Figure 1

Correlation between substrate characteristics, stem cells feedback, and the corresponding biomarker.

Figure 2

SEM images of scaffolds: (A) PCL solvent cast film (Mag 595×); (B) CNTs-reinforced PCL solvent cast film (Mag 745×); (C) PCL electrospun film (Mag 610×); and (D) CNTs-reinforced PCL electrospun film (Mag 762×).

Figure 3

Topographical AFM analysis and 3D patterns of scaffolds: (A) Roughness of PCL solvent casting scaffold on 5 × 5 scan area; (B) 3D pattern of PCL solvent casting scaffold on 5 × 5 scan area; (C) Roughness of PCL electrospun scaffold on 5 × 5 scan area; (D) 3D pattern of PCL electrospun scaffold on 5 × 5 scan area.

Figure 4

Contact angle in scaffolds before and after immersion in culture medium.

Figure 5

(a) Elasticity modulus of the films prepared by solvent casting method vs. films prepared by electrospinning and (b) maximum strength.

Figure 6

Cell proliferation: Quality MTT 3, 7, and 14 days of incubation on the different substrates.

Figure 7

Quantification of MTT levels on all substrates after 3, 7, and 14 days of incubation.

Figure 8

Levels of (a) alkaline phosphatase, (b) total protein, and (c) alkaline phosphatase per total protein in cells after 1, 3, and 7 incubation days.

Figure 9

Quality alizarin red test on 14th day and 21st day for substrates with both high and low elasticity modulus.

Figure 10

Quantity alizarin red test at 14 and 21 days of incubation on substrates.

Figure 11

The 7th and the14th day of culture: Cells’ nucleus (stained with DAPI (blue)) and Cytoskeleton Actin (stained with phalloidin (red)) on PCL solvent cast, PCL-CNTs solvent cast, PCL electrospun, P-CNTs electrospun, and titanium, respectively (magnification 10×).

Figure 12

Values of (a) contact angle (blue line), MTT (green line), and ALP/TP (orange line) for the four manufactured substrates and titanium and (b) Young’s modulus (orange line) for the four manufactured substrates together with MTT (green line) and ALP/TP (blue line).