1. Introduction
In the US alone, each year over 500,000 bone grafts are used to treat bone defects or disorders. The bone grafting products and procedures result in a burden of over $2.5 billion to the healthcare system, which is expected to double by 2020 [1]. Currently, autologous bone grafts are considered the gold standard for bone regeneration, but they are still limited as they require a second operation to harvest the bone tissue which can often result in donor site morbidity [2]. In order to avoid this, bone tissue engineering is an alternative option that combines scaffolds, cells, and bioactive factors to enhance bone repair and regeneration [3, 4].
The use of bone marrow-derived mesenchymal stem cells (BMSCs) has been a common approach for bone tissue engineering applications [5]. To improve the efficacy of stem cells used for bone tissue engineering, bioactive molecules (e.g. growth factors) are commonly incorporated with cells into a scaffolding material. Typically, growth factors such as bone morphogenic proteins (BMPs), vascular endothelial growth factors (VEGFs), and insulin-like growth factors (IGFs) have been used for bone tissue engineering. Especially, BMPs are a family of osteogenic growth factors that include BMP-2 and BMP-7 which have both been incorporated onto FDA approved devices [6]. However, growth factors for bone regeneration have been limited due to their rapid degradation in physiological conditions as well as their deactivation by enzymes [6]. A variety of both organic and inorganic nanoparticles have been utilized to overcome these challenges [7]. Most nanoparticles used for bone tissue engineering have attempted to deliver bioactive molecules such as BMP-2 [8, 9] or dexamethasone [10] to directly control differentiation fate of native stem cells or to improve bone mineral deposition, but these osteogenic molecules are still limited in their effectiveness due to their short in vivo half-life, the low loading efficiencies associated with nanoparticles [11], their toxicity and the large quantities of nanoparticles needed for therapeutic effect [6].
MicroRNAs (miRNAs) are small, non-coding RNAs that affect cell proliferation, differentiation, and apoptosis by degrading or inhibiting the translation of mRNAs [12]. Unlike other bioactive molecules, miRNAs have the target-specific nature that modulates specific genes [13]. Thus, researchers have recently been interested in delivering miRNAs associated with osteogenesis to improve osteogenic differentiation of BMSCs and therefore bone tissue regeneration [14-16]. Although bone-specific miRNAs play a critical role in bone remodeling [17], their use has been limited due to the difficulty with delivering them to target cells without interference from degrading enzymes. Some research groups have developed lipid-based [18], and even polymeric nanoparticles [19] to deliver miRNAs. Single miRNA delivery still lacks the therapeutic potential of delivering multiple miRNAs especially in bone remodeling where the miRNAs involved act to both inhibit osteoclast activity [20] as well as increasing osteoblast activity [21]. This results in a net increase in the bone mineral deposition that is much greater than the effect of delivering either miRNA alone.
To overcome these limitations, researchers have recently found that naturally derived extracellular vesicles (EVs) can be used as vesicles to deliver multiple miRNAs to target cells. EVs include exosomes, which are small nanovesicles composed of lipid membranes and are excreted by all cell types. These nanovesicles are involved in cell communication, protein transfer, and delivery of microRNAs to surrounding cells [22]. They can be easily extracted from bodily fluids or cell culture medium [22] and also have advantages such as low immunogenicity, high stability, and an intrinsic homing effect which makes them easily absorbed by target cells [23, 24], where the osteogenic miRNAs can then be delivered.
BMSC-derived EVs have been shown to play a key role in the maintenance of bone remodeling [25]. These EVs have been shown to enhance the osteogenic differentiation of BMSCs and improve bone formation depending on their miRNA profile. The miRNA profile of a stem cell-derived EV depends on the cell source [26, 27] and stage/time point of differentiation. Moreover, the use of BMSCs is limited as they are not readily abundant and require an invasive procedure to be harvested from the donor. The BMSCs are also limited in their passage capabilities, where a typical BMSC will lose its “stemness” around passage 10 [28].
Placental stem cells (PSCs) have a faster proliferation rate [28, 29] and can be expanded to larger passage numbers while still maintaining their “stemness.” PSCs have been shown to have a proliferation capacity around 30 passages which is almost triple the capacity of adult BMSCs [28]. This is due to the PSCs being closer to embryonic tissue than BMSCs which are typically harvested from adult tissue which gives them limited differentiation capabilities [28]. PSCs can also be easily harvested for autogenic or allogenic use from the postpartum placental tissue without the need for any invasive procedures associated with BMSCs [30]. Taken together, this indicates that PSCs may be an ideal candidate to isolate large quantities of EVs. PSCs have also been shown to exhibit osteogenic differentiation capabilities [31] and are widely used in tissue engineering applications due to their immunomodulation properties [32]. Although EVs from BMSCs are well characterized, the effect of osteogenic differentiation on PSC-derived EV content is poorly understood.
In this study, we aimed to compare the ability of PSC-derived EVs to differentiate with the well-characterized BMSC-derived EVs. We further aim to prove the feasibility of utilizing these EVs for bone tissue engineering applications. Since PSCs exhibit the ability to differentiate into osteogenic cell lines, we hypothesize that EVs isolated from these cells during differentiation could enhance the osteogenic differentiation of BMSCs. We evaluated the effects of cell source and stage of differentiation on the miRNA content of EVs.