Anti-Inflammatory/Antioxidant Features of Adipose Tissue Mesenchymal Stem Cells Conditioned Media for Doped TiO2 Nanoparticles in Induced Inflammation

Abstract

The fundamental purpose of this work is to determine the anti-inflammatory/antioxidant activity of stem cell conditioned medium enhanced by mono- or dual-doped TiO₂ nanoparticles. The transmission electron microscope, X-ray diffraction, and energy-dispersive X-ray analyses are used to characterize the nanoparticles. Isolation/characterization of adipose tissue mesenchymal stem cells (AD-MSCs) is done. In vitro assays reveal that protein denaturation and proteinase induction are significantly increased with lipopolysaccharide (LPS) comparable to control, while treatments significantly decrease the induction compared to LPS. In vitro assays reveal decreasing in hydroxyl radical scavenging and DPPH radical scavenging activities with LPS, while treatments significantly increase the activity compared to LPS. Induction with LPS decreases in vitro catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) expression levels and enzyme activities are significantly compared to controls, while treatments significantly increase the CAT expression compared to LPS. Induction with LPS increases the in vitro interleukin 4 (IL4), IL6, IL10, and tumor necrosis factor α expression levels, and their activities significantly decline with treatment compared to controls, while treatments significantly decline expression compared to LPS. The anti-inflammatory and antioxidant properties of AD-MSC-conditioned medium enhanced with mono- or dual-doped TiO₂ nanoparticles are identified in this investigation. To sum up, the work has shown that mono- and dual-doped TiO₂ can inhibit the inflammation caused by LPS in vitro.

Keywords: adipose tissue mesenchymal stem cells; antioxidants; doped TiO2; inflammation; lipopolysaccharide.

Figure 1

AD‐MSCs characterization. A–D) CD90, CD73, CD34, and CD45 identification of AD‐MSCs using flow cytometry. E) AD‐MSCs 3 days after isolation. F) AD‐MSCs complete sheet.

Scheme 1

Diagram for the formation of nanoparticles and the equations for the reactions that took place to obtain the nanoparticles. The obtained nanoparticles color change was TiO₂ white color, Ti0.97Cu0.03O2, and Ti0.97Cu0.015Zn0.015O2 beige color. The preparation steps we mentioned in the equation such as temperature, continuous steering, and precipitation. In addition to the accuracy in the preparation based on the protocols for nanomaterial preparations also we followed the personal protective equipment (PPE) including protective clothing, helmets, goggles, or other garments or equipment designed to protect the wearer's body from hazards.

Figure 2

XRD and bandgap of the NPs. A) XRD patterns of a) pure TiO₂, b) Ti_0.985Cu_0.015O₂, and c) Ti_0.97Cu_0.015Zn_0.015O₂ powders. B) a) diffuse reflectance and b) optical bandgap of pure TiO₂, Ti_0.97Cu_0.03O₂, and Ti_0.97Cu_0.015Zn_0.015O₂ samples.

Figure 3

TEM images of a) TiO₂, b) Ti_0.985Cu_0.015O₂, and c) Ti_0.97Cu_0.015Zn_0.015O₂ powders.

Figure 4

EDX pattern of a) pure, b) Cu doped ), and c) (Zn, Cu) codoped TiO₂ samples.

Figure 5

Cell viability of AD‐MSCs following induction with LPS, treatment with TiO₂ alone (Ti), TiO₂ mono doped with copper (Ti Mono), TiO₂ dual doped with copper and zinc (Ti Dual). a: significant difference versus CT at (p < 0.05).

Figure 6

Oxidative markers. A) MDA, B) NO, C) TAC, and D) 5‐LOP levels in CM of AD‐MSCs treated with LPS, TiO₂ to which LPS was added, mono‐doped TiO₂ to which LPS was added (Ti mono + LPS), and dual‐doped TiO₂ to which LPS was added (Ti dual + LPS). a: significant difference versus CT at (p < 0.05). b: significant difference versus LPS at (p < 0.05). c: significant difference versus LPS at (p < 0.01).

Figure 7

Oxidative modulators. A) Protein denaturation inhibition percentage, B) proteinase inhibitory percentage, C) hydroxyl radical scavenging activity, and D) DPPH inhibition percentage in CM of AD‐MSCs treated with LPS, TiO₂ to which lipopolysaccharide was added, mono‐doped TiO₂ to which LPS was added (Ti mono + LPS), and dual‐doped TiO₂ to which LPS was added (Ti dual + LPS). a: significant difference versus CT at (p < 0.05). b: significant difference versus LPS at (p < 0.05). c: significant difference versus LPS at (p < 0.01).

Figure 8

Genetic and protein expression levels of interleukins and TNF‐α. A,B) Genetic expression levels of interleukins‐4, 6 and C) TNF‐α. D,E) Protein expression levels of interleukins‐4, 6 and F) TNF‐α in CM of AD‐MSCs treated with LPS, TiO₂ to which lipopolysaccharide was added, mono‐doped TiO₂ to which LPS was added (Ti mono + LPS), and dual‐doped TiO₂ to which LPS was added (Ti dual + LPS). a: significant difference versus CT at (p < 0.05). b: significant difference versus LPS at (p < 0.05). c: significant difference versus LPS at (p < 0.01).

Figure 9

Genetic and protein expression levels of CAT, SOD, and GPx. A–C) Genetic expression levels of CAT, SOD, and GPx. D–F) Protein expression levels of CAT, SOD, and GPx in CM of AD‐MSCs treated with LPS, TiO₂ to which lipopolysaccharide was added, mono‐doped TiO₂ to which LPS was added (Ti mono + LPS), and dual‐doped TiO₂ to which LPS was added (Ti dual + LPS). a: significant difference versus CT at (p < 0.05). b: significant difference versus LPS at (p < 0.05). c: significant difference versus LPS at (p < 0.01).