Proliferation and stemness preservation of human adipose-derived stem cells by surface-modified in situ TiO₂ nanofibrous surfaces

Abstract

Two important criteria of an ideal biomaterial in the field of stem cells research are to regulate the cell proliferation without the loss of its pluripotency and to direct the differentiation into a specific cell lineage when desired. The present study describes the influence of TiO2 nanofibrous surface structures on the regulation of proliferation and stemness preservation of adipose-derived stem cells (ADSCs). TiO2 nanofiber arrays were produced in situ onto Ti-6Al-4V substrate via a thermal oxidation process and the successful fabrication of these nanostructures was confirmed by field emission scanning electron microscopy (FESEM), energy dispersive spectrometer (EDS), X-ray diffractometer (XRD), and contact angle measurement. ADSCs were seeded on two types of Ti-6Al-4V surfaces (TiO2 nanofibers and flat control), and their morphology, proliferation, and stemness expression were analyzed using FESEM, AlamarBlue assay, flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR) after 2 weeks of incubation, respectively. The results show that ADSCs exhibit better adhesion and significantly enhanced proliferation on the TiO2 nanofibrous surfaces compared to the flat control surfaces. The greater proliferation ability of TiO2 nanofibrous surfaces was further confirmed by the results of cell cycle assay. More importantly, TiO2 nanofibrous surfaces significantly upregulate the expressions of stemness markers Sox-2, Nanog3, Rex-1, and Nestin. These results demonstrate that TiO2 nanofibrous surfaces can be used to enhance cell adhesion and proliferation while simultaneously maintaining the stemness of ADSCs, thereby representing a promising approach for their potential application in the field of bone tissue engineering as well as regenerative therapies.

Keywords: nanofibers; pluripotency; stem cells; thermal oxidation; titania.

Figure 1

FESEM images of the (A) control Ti-6Al-4V substrate and the (B) fabricated TiO₂ nanofiber arrays. Note: The upper right insets show the respective water contact angle of each surface. Abbreviation: FESEM, field emission scanning electron microscopy.

Figure 2

Respective XRD pattern and EDS spectrum of the flat control sample and the as-grown TiO₂ nanofiber arrays. Abbreviations: EDS, energy dispersive spectrometer; NFs, nanofibers; R, rutile; XRD, X-ray diffractometer.

Figure 3

FESEM images show ADSCs adhered on control samples after (A) day 1, (B) day 3, (C) day 7, and (D) day 14 of culture compared to TiO₂ nanofibrous surfaces after (E) day 1, (F) day 3, (G) day 7, and (H) day 14 of culture. Note: The area highlighted by the red box is shown in higher magnification in the images (I, J, K, and L) on days 1, 3, 7, and 14, respectively. Abbreviations: ADSCs, adipose-derived stem cells; FESEM, field emission scanning electron microscopy.

Figure 4

Cell proliferation of ADSCs cultured on TiO₂ nanofibrous surfaces in comparison to the flat control sample at days 1, 3, 7, and 14. Note: Statistical significance was assessed relative to the control sample for each day (*P<0.05). Abbreviations: ADSCs, adipose-derived stem cells; NFs, nanofibers.

Figure 5

Representative DNA histogram of ADSCs cultured on control Ti-6Al-4V surfaces and TiO₂ nanofibrous surfaces. Note: The percentages of cells residing in the G0/G1 phase, S phase, and G2/M phase are shown in the histograms. Abbreviations: ADSCs, adipose-derived stem cells; NFs, nanofibers; PI-A, propidium iodide-area.

Figure 6

Relative expression of cell cycle–regulated genes: (A) CyclinD1, (B) pRb, (C) GADD45, and (D) p53, by ADSCs cultured on both the control and TiO₂ nanofibrous surfaces for 7 days and 14 days. Note: Statistical significance was assessed relative to the control sample for each time interval (*P<0.05). Abbreviations: ADSCs, adipose-derived stem cells; NFs, nanofibers.

Figure 7

Relative expression of stemness marker genes: (A) Nanog3, (B) Rex-1, (C) Sox-2, and (D) Nestin, by ADSCs cultured on both the control and TiO₂ nanofibrous surfaces for 7 days and 14 days. Note: Statistical significance was assessed relative to the control sample for each time interval (*P<0.05). Abbreviations: ADSCs, adipose-derived stem cells; NFs, nanofibers.