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.