Simple surface engineering of polydimethylsiloxane with polydopamine for stabilized mesenchymal stem cell adhesion and multipotency

Abstract

Polydimethylsiloxane (PDMS) has been extensively exploited to study stem cell physiology in the field of mechanobiology and microfluidic chips due to their transparency, low cost and ease of fabrication. However, its intrinsic high hydrophobicity renders a surface incompatible for prolonged cell adhesion and proliferation. Plasma-treated or protein-coated PDMS shows some improvement but these strategies are often short-lived with either cell aggregates formation or cell sheet dissociation. Recently, chemical functionalization of PDMS surfaces has proved to be able to stabilize long-term culture but the chemicals and procedures involved are not user- and eco-friendly. Herein, we aim to tailor greener and biocompatible PDMS surfaces by developing a one-step bio-inspired polydopamine coating strategy to stabilize long-term bone marrow stromal cell culture on PDMS substrates. Characterization of the polydopamine-coated PDMS surfaces has revealed changes in surface wettability and presence of hydroxyl and secondary amines as compared to uncoated surfaces. These changes in PDMS surface profile contribute to the stability in BMSCs adhesion, proliferation and multipotency. This simple methodology can significantly enhance the biocompatibility of PDMS-based microfluidic devices for long-term cell analysis or mechanobiological studies.

Figure 1. Experimental outline illustrating the functionalization of PDMS surfaces polydopamine to assess prolong BMSCs stability within two culture systems.

Figure 2. Effect of polydopamine concentration (0-0.100% w/v) on MSCs (A) initial adhesion, (B) proliferation and (C) adhesion stability after 2 weeks.

The actin cytoskeleton of BMSCs was stained red (rhodamine phalloidin) and the cell nucleus was stained blue (DAPI), scale bar = 200 μm. *p-value < 0.0132 showed significant differences between any two groups (Tukey HSD test). #p-value < 1 × 10⁻⁴ revealed significant difference between the groups (One way ANOVA). All data are presented as mean ± s.d. (n = 4).

Figure 3. Effect of polydopamine (0.01% w/v) coating time (0−24 h) on MSC (A) initial adhesion and (B) proliferation.

*p-value < 0.0419 shows significant differences between any two groups (Tukey HSD test). #p-value < 1.35 × 10⁻⁹ reveals significant difference between the groups (One way ANOVA). All data are presented as mean ± s.d. (n = 4).

Figure 4. Effect of polydopamine coating with collagen on MSCs (A) initial adhesion, (B) proliferation and (C) adhesion stability after 2 weeks.

The actin cytoskeleton of BMSCs was stained red (rhodamine phalloidin) and the cell nucleus was stained blue (DAPI), scale bar = 200 μm. *p-value = 0.00101 showed significant differences between any two groups (Tukey HSD test, n = 4). **p-value = 0.00175 shows significant difference between the groups (Tukey HSD test, n = 4). #p-value < 3.67 × 10⁻¹⁰ revealed significant difference between the groups (One way ANOVA). All data are presented as mean ± s.d. (n = 4).

Figure 5. Water contact angle measurement of uncoated/coated PDMS surfaces with (A) different PD concentration after 24 h, (B) different coating time of 0.010% PD and (C) PD and collagen coating.

EDS spectrum of uncoated/coated PDMS surfaces for the detection of (D) carbon and (E) nitrogen elements. (F) Ninhydrin assay for the detection of free amine group across different PD concentration coating after 24 h. *p-value < 0.00621 showed significant differences between any two groups (Tukey HSD test). **p-value = 0.00101 showed significant differences between two groups (Tukey HSD test). All data are presented as mean ± s.d. (n = 4).

Figure 6

(A) Alizarin Red staining, scale = 500 μm and well diameter = 15 mm; (B) Oil Red staining, scale bar = 500 μm; (C) gene expression of osteogenic (ALP) and adipogenic (Leptin) markers. *p-value < 0.00293 shows significant difference as compared to any other group (Tukey HSD test, n = 4). **p-value < 0.00541 shows significant difference between the groups (Tukey HSD test, n = 4), #p-value < 0.0240 shows significant difference as compared to any PD ± C1 coated PDMS (Tukey HSD test). All data are presented as mean ± s.d. (n = 4).

Figure 7

(A) F-Actin localization of cells under normal expansion, (B) Alizarin Red staining of osteogenic cultures, microfluidic chips = 22 mm × 22 mm, and (C) Oil Red staining of adipogenic cultures for 3 weeks in a microfluidic culture system. Scale bar = 500 μm.