Prolonged proliferation and delayed senescence of the adipose-derived stem cells grown on the electrospun composite nanofiber co-encapsulated with TiO₂ nanoparticles and metformin-loaded mesoporous silica nanoparticles

Abstract

This study was aimed to investigate the effects of the Poly-ε-Caprolactone/Gelatin nanofibers (PCL/GEL NFs) co-encapsulated with TiO₂ nanoparticles (nTiO₂) and metformin-loaded mesoporous silica nanoparticles (MET@MSNs) on prolonging the in vitro expansion of human adipose-derived stem cells (hADSCs) without inducing cellular senescence and aging. FTIR, BET, FE-SEM, and TEM were applied to characterize the fabricated MET@MSNs and electrospun composite NFs. The presence of inorganic particles, nTiO₂ and MSNs, in the scaffolds improved their mechanical properties and led to a more sustained release of MET with almost the lack of the initial burst release from nTiO₂/MET@MSNs-loaded NFs. The enhanced adhesion, metabolic activity, and proliferation rate of the hADSCs grown on nTiO₂/MET@MSNs-loaded NFs were demonstrated via FE-SEM images, MTT test and PicoGreen assay, respectively, over 28 days of culture. Furthermore, the irregular nanofibrillar structures and the impact of sustained release of MET led to a significant upregulation in the mRNA levels of autophagy (Atg-5, Atg-7, Atg-12, and Beclin-1) and stemness (Nanog3, Sox-2, and Oct-4) markers as well as a considerable down-regulation of p16^INK4A senescence marker. Further, the upregulation of hTERT, enhanced activity of telomerase, and increased telomere length were more pronounced in the hADSCs cultured on nTiO₂/MET@MSNs-loaded NFs as compared to other types of NFs. Overall, our findings demonstrated the potential of the fabricated nanocomposite platform for counteracting cellular senescence and achieving sufficient quantities of fresh hADSCs with preserved stemness for various stem cell-based regenerative medicine purposes.

Introduction

Adipose-derived stem cells (ADSCs) have been used in regenerative medicine in recent years and delivered more promising results than bone marrow mesenchymal stem cells (BM-MSCs). ADSCs seem to be more beneficial and potent than BM-MSCs due to their ease of isolation through slightly invasive techniques such as resection or liposuction, age-independent quality and proliferation, and the secretion of many soluble factors that mediate mobilization of endogenous stem cells (Konno et al., 2013). Besides, in exposition to appropriate growth factors and environmental conditions, ADSCs can differentiate into various cell types derived from three germ layers, similar to BM-MSCs (Mazini et al., 2019, Naderi et al., 2017). On the other hand, the use of these cells does not have the bioethical and technical limitations that apply to embryonic and pluripotent stem cells, respectively (Dadashpour et al., 2017).

Prolonged in vitro cell culturing and repeated passaging is a prerequisite to acquiring an appropriate quantity of ADSCs for use in stem cell therapy and regenerative medicine. Although, this process has a negative effect on the physiological conditions of ADSCs and leads to developing cellular senescence and aging, consequently losing their proliferative and self-renewal capacities (Deldar et al., 2017). Aging is a widely studied phenomenon characterized by some distinct cellular and biological alterations. The major cellular changes associated with aging include decreased genetic stability, altered epigenetic patterns, loss of proteostasis, nutrition stress, loss of normal mitochondrial functioning, cellular senescence, exhaustion of stem cells, dysregulated intercellular communication, and finally telomere shortening (López-Otín et al., 2013). Besides, cell aging is characterized by autophagy which markedly reduces the number and normal function of stem cells. Although autophagy is exploited by stem cells to discard unwanted cellular components during the quiescence phase, impaired autophagy can contribute to the aging and exhaustion of stem cells (Jung and Brack, 2014).

For delivering acceptable clinical outcomes in regenerative medicine, ADSCs must be abundant, young, freshly prepared and have high proliferative, differentiation, and regenerative capacities. Cellular exhaustion, senescence, loss of stemness, shortening of telomere, and reduced autophagy levels are among the most important processes leading to stem cell aging. Efforts have been made that enable ADSCs to increase their proliferation capacity in ex vivo while preserving stemness and not entering the cellular senescence and aging. Some of these efforts include pharmacological modifications, applying various culture methods, modifying cell culture media components, and using a wide range of biocompatible materials (Abdollahiyan et al., 2021a, Saei Arezoumand et al., 2017).

In fabricating the tissues of interest, nano-scaffolds are expected to provide physical support and a platform mimicking the natural features of the extracellular matrix (ECM) (Firouzi-Amandi et al., 2018). Biomimetic electrospun nanofibrous scaffolds have demonstrated effective mimicking ECM functional and structural features due to their high surface-to-volume ratio, excellent porosity, and small nanoscale diameter (Mellatyar et al., 2018, Rasouli et al., 2020). In several studies, a variety of biological substances and molecules (e.g., peptides, antimicrobial agents, small-molecule drugs, growth factors, etc.), as well as various nanoparticles (such as hydroxyapatite, TiO₂, ZnO, and AgNPs) have been incorporated into NFs to modify them in a manner that they can support cellular functions like adhesion, proliferation, and differentiation (Abdollahiyan et al., 2020, Afsharian and Rahimnejad, 2020, Khodadadi et al., 2020).

The effects of physical and mechanical stresses on MSCs are generally evaluated via culturing these cells into a soft matrix or on a flexible membrane. However, TiO₂/titanium substrates have provided an appropriate interface to assess the impact of mechanical strain on osteogenic properties MSCs in clinical settings (Chang et al., 2019). TiO₂ nanotubes with a diameter of 70 nm showed the most suitable platform for supporting the expansion of hADSCs and their differentiation to osteoblasts, suggesting the role of nanoscale geometry on the differentiation and proliferation of stem cells. Besides, it was reported that a tube diameter of 15–30 nm was appropriate to support cellular adhesion and direct BM-MSCs towards producing osteoblasts (Bauer et al., 2009, Park et al., 2007). TiO₂ nanofibrous scaffolds have been shown to appropriately bind to ADSCs and support their proliferation via inducing the expression of the genes involved in preserving the stemness, including Nestin, Rex-1, Nanog3, and Sox-2 (Tan et al., 2014). Moreover, TiO₂ nanoparticles (nTiO₂)-based nanocomposites have recently drawn considerable attention as bone substitute materials and injectable pastes to fill defects due to their excellent mechanical properties, high chemical stability and bioactivity (Rasoulianboroujeni et al., 2019).

Metformin (MET), a first-line prescription drug for type 2 diabetes mellitus, has been widely investigated as an anti-aging agent (Glossmann and Lutz, 2019, Novelle et al., 2016, Piskovatska et al., 2019, Soukas et al., 2019). MET has been shown to decrease the incidence of age-associated diseases such as Alzheimer's disease and various cancers, prevent cognitive deterioration, and reduce diabetes-related mortality (Barzilai et al., 2016). MET affects mesenchymal stem cell proliferation and differentiation and have shown a promising prospect in gerontology and stem cell-based regenerative medicine (Jiang and Liu, 2020).

Mesoporous silica nanoparticles (MSNs) are among promising candidates for delivering therapeutic molecules at a slow and sustained release rate. Unique features such as extensive surface area, numerous biologically active groups and suitable pore properties (e.g., well-shaped, appropriate size) present them as an exciting platform for efficient drug/gene delivery (Zhou et al., 2018). Incorporating drug-loaded MSNs into NFs can overcome the rapid burst drug discharge and guarantee sustained desirable release kinetics.

By modifying the polymers used for generating NFs and changing their mechanical, chemical, and surface topographical properties, it is possible to tune cellular behavior and activities (Sadeghi-Soureh et al., 2020). Therefore, in this present work, it was aimed to design and develop electrospun composite NFs co-encapsulated with nTiO₂ and MET-loaded MSNs (MET@MSNs) that provide an efficient platform for sustained and prolonged release of MET. Then, the applicability and efficiency of the NFs were evaluated to support the attachment, viability, and proliferation of hADSCs without entering them to cellular senescence and aging. An overview of our work is shown in Fig. 1.