Engineering exosomes for bone defect repair

Abstract

Currently, bone defect repair is still an intractable clinical problem. Numerous treatments have been performed, but their clinical results are unsatisfactory. As a key element of cell-free therapy, exosome is becoming a promising tool of bone regeneration in recent decades, because of its promoting osteogenesis and osteogenic differentiation function in vivo and in vitro. However, low yield, weak activity, inefficient targeting ability, and unpredictable side effects of natural exosomes have limited the clinical application. To overcome the weakness, various approaches have been applied to produce engineering exosomes by regulating their production and function at present. In this review, we will focus on the engineering exosomes for bone defect repair. By summarizing the exosomal cargos affecting osteogenesis, the strategies of engineering exosomes and properties of exosome-integrated biomaterials, this work will provide novel insights into exploring advanced engineering exosome-based cell-free therapy for bone defect repair.

Full text

Engineering exosomes for bone
defect repair
Shaoyang Ma, Yuchen Zhang, Sijia Li, Ang Li, Ye Li* and
Dandan Pei*
Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of
Stomatology, Xi’an Jiaotong University, Xi’an, Shaanxi, China
Currently, bone defect repair is still an intractable clinical problem. Numerous
treatments have been performed, but their clinical results are unsatisfactory. As
a key element of cell-free therapy, exosome is becoming a promising tool of
bone regeneration in recent decades, because of its promoting osteogenesis
and osteogenic differentiation function in vivo and in vitro. However, low yield,
weak activity, inefficient targeting ability, and unpredictable side effects of
natural exosomes have limited the clinical application. To overcome the
weakness, various approaches have been applied to produce engineering
exosomes by regulating their production and function at present. In this
review, we will focus on the engineering exosomes for bone defect repair.
By summarizing the exosomal cargos affecting osteogenesis, the strategies of
engineering exosomes and properties of exosome-integrated biomaterials, this
work will provide novel insights into exploring advanced engineering exosome-
based cell-free therapy for bone defect repair.
KEYWORDS
bone regeneration, engineering exosomes, exosomal cargos, exosome-integrated
biomaterials, osteogenesis
1 Introduction
Bone is the central element in skeletal tissues of the human body, and provides a
framework for attachment of muscles and other tissues, enables body movements, provides
protection of internal organs from injury, promotes blood cells production, and balances
calcium and acid/base homeostasis (Elefteriou, 2018). However, the regeneration of critical-
size bone defects is still a major clinical challenge and globally costs up to $45 billion per year
(Mauffrey et al., 2015;Bharadwaz and Jayasuriya, 2020). Recently, stem cell therapy is
considered as a potential strategy for bone defect regeneration (TanSHS.etal.,2020), and
several clinical studies have demonstrated mesenchymal stromal/stem cells (MSCs) to be safe
and efficacious for the treatment of bone defects and diseases (Liebergall et al., 2013;Chen
et al., 2016;Castillo-Cardiel et al., 2017;Hernigou et al., 2018a;Hernigou et al., 2018b).
Nevertheless, cellular therapies incur significant costs and challenges as they require
stringently monitored manufacturing, handling, and storage to ensure optimal viability
and potency of cells needed for transplantation (Tan et al., 2021). More importantly,
accumulating evidence indicates that the positive effect of MSCs on tissue repair is to
stimulate the activity of tissue-resident recipient cells through paracrine, such as exosomes,
OPEN ACCESS
EDITED BY
Jianxun Ding,
Changchun Institute of Applied
Chemistry (CAS), China
REVIEWED BY
Shiyu Liu,
Fourth Military Medical University, China
Qianli Ma,
University of Oslo, Norway
Mingming Yang,
Northwest A&F University, China
*CORRESPONDENCE
Ye Li,
lisa.l0309@163.com
Dandan Pei,
peidandan@xjtu.edu.cn
SPECIALTY SECTION
This article was submitted
to Biomaterials,
a section of the journal
Frontiers in Bioengineering and
Biotechnology
RECEIVED 07 November 2022
ACCEPTED 28 November 2022
PUBLISHED 07 December 2022
CITATION
Ma S, Zhang Y, Li S, Li A, Li Y and Pei D
(2022), Engineering exosomes for bone
defect repair.
Front. Bioeng. Biotechnol. 10:1091360.
doi: 10.3389/fbioe.2022.1091360
COPYRIGHT
© 2022 Ma, Zhang, Li, Li, Li and Pei. This
is an open-access article distributed
under the terms of the Creative
Commons Attribution License (CC BY).
The use, distribution or reproduction in
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original author(s) and the copyright
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or reproduction is permitted which does
not comply with these terms.
Frontiers in Bioengineering and Biotechnology frontiersin.org01
TYPE Review
PUBLISHED 07 December 2022
DOI 10.3389/fbioe.2022.1091360

rather than directly differentiate into parenchymal cells to repair or
replace damaged tissue (Liang et al., 2014;Zhang et al., 2016). Such
concerns have driven the search for alternate therapeutic strategies
and cell-free therapies based on exosomes have become strongly
established in the landscape of regenerative medicine.
Exosome is a subclass of membrane-coated extracellular
vesicles with sizes of 30–150 nm (Tkach and Thery, 2016). As
one of the most revolutionary contributions to cell biology in the
past 30 years (Wang Y. et al., 2019), exosomes can exert multiple
biological functions by targeting recipient cells and inducing
signaling via receptor-ligand interactions, endocytosis and/or
phagocytosis (Bobrie et al., 2012;Colombo et al., 2014;
Hoshino et al., 2015;Shang et al., 2021). Exosomes have been
experimented with many animal models for the regeneration of
bone, osteochondral, and cartilage injury/diseases such as
osteoarthritis (OA), osteoporosis, osteonecrosis, and
inflammatory bone loss in periodontitis with enhanced tissue
formation and integration (Kuang et al., 2019;Kim et al., 2020;
Lei et al., 2022). Furthermore, several exosome-based clinical
experiments of orthopedic diseases have been performed based
on US-NIH clinical trial database (https://clinicaltrials.gov/).
However, there still are several constraints to exosome clinical
applications for bone defect repair: 1) unclear mechanism of
promoting bone tissue regeneration; 2) poor retention and
targeting ability of exosome at the bone defect site; 3) low
extraction rate and complex separation process.
In view of the shortcomings of natural exosomes, a growing
number of studies are aiming to develop engineering exosomes
based on modifying exosomal cargos or/and incorporating
biomaterials (Bei et al., 2021;Lathwal et al., 2021;Liang et al.,
2021). Here we will review the recent research of engineering
exosome used in bone defect repair, and highlight the bioactive
cargos and construction strategies. Additionally, we will also
summarize the application of biomaterials to impregnate
exosome and focus on how the properties of biomaterials
assist exosome to promote bone regeneration. By reviewing
currently available knowledge, this present review will
contribute to the clinical knowledge and may have
implications for the engineering design of exosomes used in
bone defect repair.
2 Osteogenic cargos in exosomes
In the past decade, numerous exosomal bioactive cargos have
been revealed (Kalluri and LeBleu, 2020). Exosomal cargos are
dependent on the parent cell type and vary between different
physiological or pathological conditions (Meng et al., 2019). The
vesicular structure of exosome provides an enclosed space to
protect exosomal cargos against degradation. In return, exosomal
cargos are the foundation to endow exosomes with various
biological functions. In this section we will review recent
research about exosomes in bone regeneration and focus on
the functions of exosomal cargos and their molecular
mechanisms (Figure 1).
2.1 Non-Coding RNA
Non-coding RNAs (ncRNAs) refer to the RNAs that lack
protein-coding regions, and have the potential to regulate gene
expression at transcriptional, post-transcriptional, and
translational levels, thereby modulating associated signaling
networks (Bhat et al., 2020). NcRNAs have become a hot
topic of increasing concern after the completion of the
Human Genome Project (Lander et al., 2001), which showed
only 1.2% of genes in the genome could encode proteins, whereas
the rest were considered as “non-coding”. Accumulating
evidence demonstrates that a variety of ncRNAs can be
encapsulated and transported by exosomes, among which
exosomal microRNAs (miRNAs), long non-coding RNAs
(lncRNAs), and circular RNAs (circRNAs) are the most
attractive subclasses in the field of bone regeneration.
2.1.1 miRNAs
MiRNAs are small, highly conserved ncRNAs with ~22 nt
length (Prattichizzo et al., 2021). The biogenesis of miRNAs
involves the processing of larger primary miRNAs (pri-miRNAs)
into shorter pre-miRNAs, and the maturation of pre-miRNA to
produce active miRNAs (Ha and Kim, 2014). MiRNAs mediate
post-transcriptional gene silencing by binding to the target
mRNAs 3′-untranslated region (UTR) or open reading frames
(ORFs) to regulate the translational process in a wide range of
physiological processes (Yang et al., 2017).
Since the first observation of exosomal miRNAs in 2007
(Valadi et al., 2007), miRNAs have become the most studied
cargos in exosome. Recently, a massive number of studies have
demonstrated that miRNAs in natural exosomes derived from
multiple cell types can promote osteogenesis (Table 1). These
studies confirmed miRNAs from exosomes of different cellular
origin can enter recipient cells with the help of exosome
internalization, and then regulate the expressions of genes
associated with osteogenic at the translational level to regulate
bone regeneration.
2.1.2 lncRNAs
As a heterogeneous group of non-protein-coding
transcripts with length of greater than 200 nucleotides,
lncRNAs are emerging regulators involved in diverse
physiological and pathological processes (Kopp and
Mendell, 2018;Nair et al., 2020). Notably, lncRNAs can be
selectively packaged into exosomes (Valadi et al., 2007), which
enable them as biomarkers of certain disease. For instance, the
expressions of lncRNAs in serum exosomes from persons with
or without osteoporosis showed significant differences (Teng
et al., 2020).
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Beyond as molecular markers, lncRNAs can sponge miRNAs
and regulate the expression of downstream genes, called
competing endogenous RNA (ceRNA) mechanism (Salmena
et al., 2011). Accumulating evidence showed that lncRNAs
from multiple cells-derived exosomes can enter the receptor
cells and have the potential to regulate bone regeneration
(Table 1).
2.1.3 circRNAs
CircRNA, a special subclass of lncRNAs with a circular
structure, has recently gained interest because of their
extraordinary stability, much longer half-life and diverse
biological functions (Jeck and Sharpless, 2014;Liu and Chen,
2022). CircRNAs can be selectively packaged into exosomes
similar to lncRNAs (Ma et al., 2021). Additionally, exosomal
circRNA also has the potential to regulate gene expression by
ceRNA mechanism (Zhi et al., 2021;Du et al., 2022). Number of
studies have revealed the regulatory function of exosomal
circRNA in bone regeneration (Table 1). Interestingly, the
effect of exosomal circRNAs in regulating bone regeneration,
it seems, is a double-edged sword. For example, Zhi et al. (2021)
reported that serum exosomal hsa_circ_0006859 was
upregulated in patients with osteoporosis, and suppressed
osteogenesis and promoted adipogenesis. Therefore, the
regulatory functions of exosomal circRNAs are still unclear,
which needs further studies.
2.1.4 tsRNAs
Transfer RNA (tRNA)-derived small RNA (tsRNA) is a class
of small ncRNAs generated from precursor or mature tRNAs,
which has recently received considerable attention (Zhu et al.,
2018;Zhu et al., 2019). With the deepening of research, tsRNAs
have been reported to regulate stem cell maintenance (Blanco
et al., 2016), cancer (Balatti et al., 2017), viral infection (Nunes
et al., 2020), neurological diseases (Zhang et al., 2020), epigenetic
inheritance (Zhang Y. et al., 2018), and symbiosis (Ren et al.,
2019). The mechanisms of action of tsRNAs include playing as
mimicry/replacement of tRNAs with sequence/structure effects,
associating with ribonucleoproteins and binding to the target
genes like miRNAs (Chen et al., 2021). Although the function of
exosomal tsRNAs is an emerging field with a paucity of research,
Fang et al. (2020) explored the osteogenic effect of exosomal
tsRNA (Table 1). They found tsRNA-10277 in the exosome
derived from BMSCs could enhance osteogenic differentiation
ability of dexamethasone-induced BMSCs.
2.2 mRNAs
As the Central Dogma of molecular biology presented
mRNA as the fundamental ingredient in genetic translational
machinery (Crick, 1970), it seemed that transferring mRNA via
exosomes to affect the biological processes of recipient cells
would be a more simple and efficient method compared with
transferring ncRNAs. However, there has been remarkably little
work about exosomal mRNA. This is probably because miRNAs
and lncRNAs are the vast majority of exosomal RNAs
(Hergenreider et al., 2012;Zhang et al., 2015;Zhang et al.,
2017), and exosomal mRNAs were classically thought to be in
the form of fragments, but not their intact forms (Valadi et al.,
2007;Wei et al., 2017). With further research, it was estimated
that on average, one intact mRNA can be found within every
1,000 exosomes produced endogenously without external
stimulation (Yang Z. et al., 2020). Therefore, it is essential to
confirm the integrity, high expression and regulatory function of
mRNAs in the research based on exosomal mRNAs. In recent
FIGURE 1
Schematic of exosomal cargos (miRNA, lncRNA, circRNA, tsRNA, mRNA and protein) with the function of promoting bone regeneration.
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TABLE 1 The exosomal cargos involved in bone regeneration.
Cargos Sources Target cell Function References
ncRNA
miRNA
miR-23a-3p UCMSCs Chondrocytes BMSCs Promoting the migration, proliferation and differentiation of
chondrocytes and BMSCs
Hu et al. (2020)
miR-21 UCMSCs EPCs Enhancing angiogenesis Zhang et al.
(2021c)
miR-378a M2 polarized
macrophages
MSCs Inducing osteogenic differentiation Kang et al. (2020)
miR-100-5p IPFP-MSCs Chondrocytes Enhancing the autophagy level of chondrocytes Wu et al. (2019)
miR-335 Mature DCs BMSCs Promoting the proliferation and osteogenic differentiation of BMSCs Cao et al. (2021)
miR-126 EPCs Endothelial cells Enhancing the proliferation, migration, and angiogenic capacity of
endothelial cells
Jia et al. (2019)
miR-451a ADSCs Macrophages Inhibiting inflammation and promoting the polarization of
M1 macrophages to M2 macrophages
Li et al. (2022)
miR-126–5p SCAP HUVECs Promoting angiogenesis Jing et al. (2022)
miR-150–5p MC3T3-E1 Promoting osteogenesis
miR-29a BMSCs HUVECs Promoting angiogenesis Lu et al. (2020)
lncRNA
NEAT1 Prostate cancer cells BMSCs Inducing osteogenic differentiation Mo et al. (2021)
MALAT1 EPCs Bone marrow-derived
macrophages
Enhancing recruitment and differentiation of osteoclast precursors Cui et al. (2019)
MALAT1 BMSCs hFOB1.19 Enhancing osteoblast activity Yang et al. (2019)
MEG-3 BMSCs Chondrocytes Reducing senescence and apoptosis Jin et al. (2021)
LYRM4-AS1 BMSCs Chondrocytes Regulating the growth of chondrocytes Wang et al.
(2021c)
H19 BMSCs CD31
+
ECs and BMSCs Promoting endothelial angiogenesis and BMSCs osteogenesis Behera et al.
(2021)
BMSCs Affecting osteogenic differentiation Wang et al.
(2021d)
circRNA
circLPAR1 Osteogenic-induced
DPSCs
DPSCs Promoting osteogenic differentiation of the recipient DPSCs Xie et al. (2020)
circRNA_0001236 BMSCs BMSCs Promoting chondrogenic differentiation Mao et al. (2021)
circ_003564 BMSCs Primary neurons and PC-12
cells
Attenuating inflammasome-related pyroptosis Zhao et al. (2022)
circ-Rtn4 BMSCs MC3T3-E1 cells Attenuating TNF-α-induced cytotoxicity and apoptosis Cao et al. (2020)
circ_0008542 MC3T3-E1 cells Osteoclast Promoting osteoclast differentiation and bone resorption Wang et al.
(2021b)
circRNA3503 SMSCs Chondrocytes Promoting chondrocyte renewal to alleviate the progressive loss of
chondrocytes
Tao et al. (2021)
cirHmbox1 Osteoclasts Osteoclasts and osteoblasts Regulating osteoclasts differentiation and osteoblasts differentiation Liu et al. (2020)
tsRNA
tsRNA-10277 BMSCs Dexamethasone-induced
BMSCs
Enhancing osteogenic differentiation ability Fang et al. (2020)
mRNA
TFAM SHED DPSCs Promoting osteogenic differentiation Guo et al. (2022)
IL-10 M2 polarized
macrophages
BMSCs Regulating cell differentiation and bone metabolism Chen et al. (2022)
Protein
CD73 MSCs Chondrocytes Suppressing inflammation and restoring matrix homeostasis Zhang et al.
(2019)
Wnt-3a ADSCs Primary osteoblastic cells Promoting the proliferation and osteogenic differentiation Lu et al. (2017)
(Continued on following page)
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research, the regulatory function of exosomal mRNA in bone
regeneration have been revealed (Table 1). These studies showed
exosomal mRNAs also could be a useful tool to aid the healing of
bone defects, as long as improving the loading efficiency of
intrinsically encapsulate transcribed mRNA into secreted
exosomes.
2.3 Protein
A variety of proteins have been observed in exosomes,
including cytoskeletal proteins, tetraspanins (CD9, CD63,
CD81, and CD82), ESCRT-associated components (Alix and
TSG101), heat shock proteins (HSP60, HSP70, and HSP90),
antigen presentation proteins (MHC I and MHC II), and
integrins (Kalluri and LeBleu, 2020;Zhu et al., 2020). As the
main executor of life activities, proteins are not only the markers
of exosomes but also endow exosomes with many biofunctions
including regulating bone regeneration (Table 1).
Despite above research drawn inspiring conclusions, the
controversy about the function of exosomal protein in bone
regeneration persists. Take BMP2, an important regulator of
osteogenesis, as an example. Han L. et al. (2022) reported that
BMP2 in BMSC-derived exosomes could promote tendon bone
healing in rotator cuff tear by activating Smad/RUNX2 signaling
pathway. Conversely, in another study, exosomes derived from
MSCs overexpressing BMP2 did not contain BMP2 protein, and
the function of promoting bone regeneration was possibly due to
the changes of exosomal miRNA composition (Huang et al.,
2020). Additionally, Furuta et al. (2016) found MSC exosomes
could promote mice fracture healing, but the levels of SDF-1,
MCP-1, and MCP-3, essential factors in the initial phase of
fracture healing (Kitaori et al., 2009;Toupadakis et al., 2012;
Ando et al., 2014;Ishikawa et al., 2014), in MSC exosomes were
significantly lower, suggesting that bone regeneration may be
mediated by other exosome components (such as miRNAs) but
not exosomal proteins. The controversy above suggests that the
mechanisms by which exosomal proteins work may be complex
and remain to be determined.
To sum up, the function of various exosomal cargos makes
exosomes have the ability to regulate bone regeneration in
different ways. Predictably, more and more exosomal cargos
would be revealed to function in bone regeneration by
conventional or novel mechanism in the near future.
Meanwhile, the explorations of mechanism inspired
investigators to design engineering exosomes for bone
regeneration by modifying the exosomal cargos, which will be
discussed in the next section.
3 Strategies of engineering exosomes
for bone defect repair
Although numerous exosomal cargos have been revealed to
function in promoting osteogenic differentiation in the past
decade, the clinical application of exosome in bone
regeneration is still facing major challenges. The reason may
be the low exosome yield, low content of functional exosomal
cargos and low targeting efficiency of native exosomes (Song
et al., 2022).
To improve the yield of exosomes, it is necessary to simplify the
exosome extraction procedure. Until now, six classes of exosome
separation strategies have been reported, including ultra-speed
centrifugation, ultrafiltration, immunoaffinity capture, charge
neutralization-based polymer precipitation, size-exclusion
chromatograph, and microfluidic techniques, with unique sets of
advantages and disadvantages for each technique (Yang D. et al.,
2020). These rapid development in separation technology has in a
large extent solved the problem of exosome isolation.
In order to enrich the exosomal cargo and increase exosome
targeting efficiency, engineering exosome is rapidly expanding in the
past decade. Engineering exosomes are the exosomes created
through changing parent cells or directly on exosomes by
biochemical or physical treatment (Kojima et al., 2018;Yerneni
et al., 2019). In this section, we summarized the three strategies of
engineering exosomes for bone regeneration (Figure 2): 1) direct
modification of exosomes, 2) chemical or physical treatment of
parent cells, and 3) genetic modification of parent cells.
3.1 Direct modification of exosomes
The direct modification of exosomes means decorating the
surface proteins to improve the targeting ability of exosomes; or
TABLE 1 (Continued) The exosomal cargos involved in bone regeneration.
Cargos Sources Target cell Function References
Mutant HIF-1αBMSCs BMSCs Promoting osteogenic differentiation capacity and angiogenesis Li et al. (2017)
HUVECs
BMP2 BMSCs BMSCs Promoting tendon bone healing in rotator cuff tear Han et al. (2022a)
UCMSCs, umbilical cord-derived mesenchymal stem cells; IPFP-MSCs, infrapatellar fat pad mesenchymal stem cells; DCs, dendritic cells; EPCs, endothelial progenitor cells; ADSCs,
adipose-derived stem cells; SCAP, stem cells from apical papilla; BMSCs, bone marrow mesenchymal stem cells; ECs, endothelial cells; DPSCs, dental pulp stem cells; SMSCs, synovium
mesenchymal stem cells; SHED, stem cells from human exfoliated deciduous teeth; DPSCs, dental pulp stem cells; MSCs, mesenchymal stem cells; HUVECs, human umbilical vein
endothelial cells.
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embellishing exosomal cargos or exogenous bioactive molecules
to enhance the regulatory function through chemical methods
(conjugation of peptides to exosomal surface (Gao X. et al.,
2018)) or physical methods (electroporation (Tian et al., 2014)
or sonication (Wang P. et al., 2019)) directly. This strategy has
been extensively used to enhance the targeting ability and/or
deliver specific cargo to the lesion region in numerous diseases,
such as cancers (Gilligan and Dwyer, 2017;Zhang and Yu, 2019;
Zhou et al., 2021), acute lung injury/acute respiratory distress
syndrome (Zoulikha et al., 2022), inflammatory bowel disease
(Ocansey et al., 2020) and Alzheimer’s disease (Alvarez-Erviti
et al., 2011).
In bone regeneration, several studies have revealed the enhanced
function of exosomes modified by electroporation and sonication.
For example, Wang et al. (2021e) used electroporation to introduce
activating transcription factor 4 (ATF4)mRNAintomiceserum
exosomes, and found the ATF4-overloading exosomes could
promote chondrocyte autophagy and inhibit chondrocyte
apoptosis, which in turn protected cartilage and alleviated
osteoarthritic progression. Zha et al. (2021) encapsulated plasmid
carrying the vascular endothelial growth factor (VEGF)into
exosomes via electroporation, and the gene-activated engineering
exosomes could effectively induce the bulk of vascularized bone
regeneration. Choi et al. (2019) inactivated pre-osteoblast exosomal
let-7, a critical miRNA regulating osteogenesis regulation, by
transfecting let-7 inhibitor into exosomes via electroporation, and
found these exosomes lost the ability to recover osteogenic
differentiation, which confirmed the availability of direct
modification of exosomes strategy from the opposite.
Additionally, although data are scarce, sonication is another
method to load hydrophilic molecules into exosomes, which is
considered much more efficient than electroporation (Kim et al.,
2016). In several studies, the mixture of BMP2 protein and exosomes
was sonicated on ice to construct BMP2-loaded exosomes (Haney
et al., 2015;Yerneni et al., 2021;Yerneni et al., 2022), and these
engineering exosomes could enhance the osteogenic potential of
MC3T3-E1 cells (Yerneni et al., 2022).
Direct modification of exosomes seems a simple and useful
approach to obtain engineering exosomes, but the application of
this strategy is still facing several challenges. The loading
efficiency of electroporation is largely suppressed when
transferring oligonucleotides with more than 750 bp length
into exosomes (Lamichhane et al., 2015). Another important
point to consider is that sonication is reported to be the most
damaging technique for exosomal membrane integrity (Donoso-
Quezada et al., 2020). Besides, the size and zeta potential were
reported to affect the efficiency of exosome internalization
(Caponnetto et al., 2017;Patel et al., 2019), which should be
taken into consideration in the further research. Therefore, when
using this strategy to product engineering exosomes, it must be
carefully designed to increase loading and internalization
efficiency and avoid exosome rupture.
3.2 Chemical or physical treatment of
parent cells
Directly treating parent cells with chemical or physical factors is
an available strategy for engineering exosomes. As originated from
parent cells, the characteristics of exosomes will be reflected by the
FIGURE 2
Three strategies of engineering exosomes for bone regeneration. ODM: osteogenic differentiation medium.
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physiological and biochemical alterations of parent cells. Numerous
studies have confirmed that the preconditioning of stem cells via
hypoxia, pharmacological agents, chemical agents, trophic factors,
cytokines, and physical factors could improve stem cells’function
in vitro and in vivo (Liu et al., 2011;Ferrer et al., 2013;Yang et al.,
2016;Kheirandish et al., 2017;Yin et al., 2017;Hu and Li, 2018).
Chemical agents and metal ions are the two main treatment
modalities of producing engineering exosomes by chemical
treatment. Culturing parent cells in the osteogenic differentiation
medium (ODM) is the most common method. Liu A. et al. (2021)
isolated exosomes from BMSCs after osteoinductive culturing and
found these engineering exosomes enhanced the bone forming
capacity and induced rapid initiation of bone regeneration. In
other research, umbilical cord mesenchymal stem cells (Ge and
Wang, 2021) and dental pulp stem cells (Xie et al., 2020)were
cultured in the ODM to produce engineering exosomes, which could
enhance osteogenesis. Besides the ODM, many other chemical
agents, TNF-α(Lu et al., 2017), short peptide (Zhao W. et al.,
2021), parathyroid hormone (Shao et al., 2022),
dimethyloxalylglycine (Liang et al., 2019)andBMP2(Wei et al.,
2019), were also used to produce engineering exosomes for bone
defect repair. The metal ions treatment of parent cells can also
endow exosomes with the ability to enhance bone regeneration. The
exosomes derived from BMSCs stimulated by strontium-substituted
calcium silicate ceramics could regulate osteogenesis and
angiogenesis of human umbilical vein endothelial cells (Liu et al.,
2021c). Similarly, macrophage-derived exosomes upon stimulation
with titania nanotubes simultaneously enhanced osteogenesis and
angiogenesis (Wang et al., 2022d).
Moreover, various physical modifications of parent cells also
could yield engineering exosomes. Wu et al. (2021a) collected
exosomes from BMSCs stimulated by magnetic nanoparticles and
a static magnetic field and found these exosomes could improve
osteogenesis and angiogenesis. As oxygen concentration plays a
crucial role in proliferation (Silván et al., 2009), Shen et al. (2022)
found exosomes derived from hypoxia preconditioned MSCs
promote cartilage regeneration via the miR-205–5p/PTEN/AKT
pathway. The mechanical force is an essential factor to regulate
the differentiation of stem cells (Halder et al., 2012). Lv et al. (2020)
found exosomes derived from osteocyte induced by mechanical
strain could promote the proliferation and osteogenic differentiation
of human periodontal ligament stem cell. Low yield is one of the
main challenges for the application of engineering exosomes. To
overcome this, Fan et al. (2020) employed an extrusion approach to
amass exosome mimetics (EMs) from human MSCs, and the EMs
demonstrated robust bone regeneration. In other studies, low-
intensity pulsed ultrasound not only promoted BMSC-exosome
release, but enhances the effects of BMSC-exosomes on cartilage
regeneration in osteoarthritis (Liao et al., 2021;Xia et al., 2022).
According to above research, chemical or physical treatment of
parent cells indeed is an effective strategy to produce engineering
exosomes for bone regeneration. It is worthwhile to mention that the
function of the engineering exosomes produced by this strategy still
relies on the exosomal cargos in substantially all these studies.
Therefore, modifying the nucleic acids of parent cells to produce
engineering exosomes with bioactive cargos seems another ideal
strategy, which will be elaborated on below.
3.3 Genetic modification of parent cells
With advances in molecular biological techniques, gene-
editing has become one of the most commonly used
methodologies in molecular research. Consequently,
FIGURE 3
The properties of biomaterial (hydrogel and metal scaffold) help exosomes to promote bone regeneration.
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engineering exosomes with more or totally new bioactive
molecules can be performed by editing certain genes in parent
cells. As described previously, cargos are the basis of exosomes
function, and numerous exosomal cargos (mRNAs, miRNAs,
lncRNAs, circRNAs and proteins) have been confirmed to
promote bone regeneration, which enlightened researchers to
produce engineering exosomes by genetic modification of the
parent cells.
As the most extensively studied exosomal cargos, miRNAs with
the function of promoting bone regeneration received tremendous
attention, and a vast variety of studies have attempted to enhance the
biofunction of exosomes by gene-editing of parent cells’miRNAs.
Wang N. et al. (2022) transfected BMSCs with lentivirus to obtain
exosomes overexpressing miR-140–3p, and found these exosomes
promoted bone defect remodeling. A lentiviral infection system was
also used to overexpress miR-940 in MDA-MB-231 cells to attain
engineering exosomes, which could promote the osteogenic
differentiation of human MSCs (Hashimoto et al., 2018).
Transferring parent cells with miRNAs by Lipofectamine reagent
is another genetic modification method. By this way, exosomes
overexpressing miR-378 (Nan et al., 2021), miR-181b (Liu W. et al.,
2021)andmiR-122–5p (Liao et al., 2019) have been demonstrated to
promote osteogenic differentiation.
The mRNA is also an important target for this strategy. Li
et al. (2017) transfected adenovirus carrying mutant HIF-1αinto
BMSCs, and found the mutant protein was highly expressed in
BMSCs exosomes, which markedly accelerated the bone
regeneration and angiogenesis. Interestingly, in several other
TABLE 2 The properties of exosome-integrated biomaterials for bone defect repair.
Properties Biomaterials References
Maintaining exosomes stability Nanocomposite hydrogels Li et al. (2021b)
Enhancing local concentrations of exosomes Acellular extracellular matrix hydrogel Xing et al. (2021)
Injectable hyaluronic acid hydrogel Zhang et al. (2021c)
Gelatin methacrylate/nanoclay hydrogel Hu et al. (2020)
3D matrix hydrogels Holkar et al. (2021)
Injectable thermosensitive hydrogel Ma et al. (2022)
Enhancing exosomes activity 3D matrix hydrogels Yu et al. (2022b)
Alginate hydrogel Holkar et al. (2021)
Optimizing the 3D distribution ABM/P-15 CMC-hydrogel Matos et al. (2012)
3D-printed porous bone scaffolds Zha et al. (2021)
Antibacterial property Food-grade probiotic-modified implant Tan et al. (2020a)
Multifunctional HA hydrogel Liu et al. (2022)
Yu et al. (2022a)
Natural polymer HA hydrogel Mi et al. (2022)
Chitosan hydrogel Shen et al. (2020)
Adapting to irregular bone defects Injectable thermosensitive hydrogel Xing et al. (2021)
PDLLA-PEG-PDLLA triblock copolymer gels Tao et al. (2021)
Chitosan hydrogel Fan et al. (2020)
Wu et al. (2021b)
Nanocomposite hydrogel based on gelatin and Laponite Liu et al. (2021b)
PG/TCP Zhang et al. (2021a)
HA hydrogel Yu et al. (2022a)
Alginate Huang et al. (2020)
Holkar et al. (2021)
Gel-ADH Lin et al. (2022)
Silk fibroin Shen et al. (2022)
Hyaluronic acid Sang et al. (2022)
Yang et al. (2020b)
SIS-CA hydrogel Ma et al. (2022)
Favorable adhesion Crosslinked network of alginate-dopamine, chondroitin sulfate, and regenerated silk fibroin Zhang et al. (2021b)
HA hydrogel modified with the PPFLMLLKGSTR peptide Li et al. (2020)
Thermo-sensitivity SIS-CA hydrogel Ma et al. (2022)
ABM/P-15, CMC-hydrogel: bovine-derived mineral bound to a P-15 carboxymethyl cellulose-hydrogel; HA, hyaluronic acid; PDLLA-PEG-PDLLA, poly (D,L-lactide)-b-poly (ethylene
glycol)-b-poly (D, L-lactide); PG/TCP, poly ethylene glycol maleate citrate with β-TCP; Gel-ADH, hydrazide grafted gelatin; SIS-CA, small intestinal submucosa with propionic acid.
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studies, exosomes derived from parent cells with gene-editing of
BMP2 (Huang et al., 2020), Scx (Feng et al., 2021) and P2X7R (Xu
et al., 2020) performed the enhanced osteogenic ability. However,
this modulating function was due to the changes of exosomal
miRNA rather than the transfection of these genes. This might be
due to two reasons: firstly, the cellular components are selectively
packaged into exosomes to be exosomal cargos (Ma et al., 2021),
and gene-editing of certain genes may not inevitably result in
their expression change in exosome; secondly, miRNAs are the
most abundant exosomal cargos, which may be more sensitive to
the gene-editing modification.
Several studies revealed the effect of genetic modification on
other exosomal bioactive molecules (lncRNAs and circRNAs). Cui
et al. (2019) inhibited lncRNA-MALAT1 expression in endothelial
progenitor cells-derived exosomes by transfecting lncRNA-
MALAT1-targeting siRNA, which disrupted bone regeneration.
Cao et al. (2020) subcloned the full sequence of circ-Rtn4 into
the pcDNA-3.1 vector and transfected the vector into BMSCs using
Lipofectamine 2000 to overexpress exosomal circ-Rtn4.
Nevertheless, to date, the research in this field has remained
limited for the technical reason. Take upregulating circRNAs as
an example, it is difficult to deplete or generate the circular form
without affecting the linear counterpart of circRNA (Nielsen et al.,
2022). In addition, low cyclization efficacy and accuracy also limited
the modification of circRNAs by gene-editing. Therefore,
investigation into novel and high-efficiency genetic modification
technologies is required to combat these problems.
4 Properties of exosome-integrated
biomaterials essential for bone defect
repair
Although the strategies of engineering exosomes could
enhance exosome yield and biofunction, exosomes used for
clinical bone defect treatment are still limited (van der Meel
et al., 2014;Lener et al., 2015). Currently, the major modes of
exosome application are direct injection or carrier loading, which
is mainly aimed at systemic diseases, such as osteoporosis (Song
et al., 2019), hematological malignancies (De Luca et al., 2017),
and myocardial ischemia-reperfusion injury (Zhao et al., 2019).
Nevertheless, it has been reported that no significant effect was
observed with free exosomes treatment by direct injection,
because of its rapid excretion from the site of application
(Zhang Z. et al., 2018;Wang C. et al., 2019;Xing et al., 2022),
suggeting the medand for exosome-integrated biomaterials.
Currently, more and more biomaterials have been designed
and applied in bone regeneration (Cui et al., 2020;Zhao D.
et al., 2021;Zhu et al., 2022a;Zhu et al., 2022b;Wang et al., 2022c;
Zhang et al., 2022). Therefore, the selection of available
biomaterials with appropriate stability and integrity to load
and release exosomes at the bone defect site to increase their
retention and stability may be necessary for bone regeneration.
Several excellent and informative reviews have addressed the
types, synthetic procedure and/or encapsulation approaches of
biomaterials used to carry exosomes (Riau et al., 2019;Pishavar
et al., 2021;Wang D. et al., 2022;Sun et al., 2022). Instead, we
propose to streamline the properties of biomaterial to dissect how
and by what mechanisms the biomaterials help exosomes to
promote bone regeneration (Figure 3;Table 2). By summarizing
the previous studies, we expected to represent a promising
strategy for the use of engineering exosomes in combination
with biomaterials for clinical bone regeneration.
4.1 Maintaining the exosome stability
The first consideration is how to maintain the stability of
exosomes. Despite the bilayer membrane structures making
exosomes resist degradation to some extent, exosomes are
unstable and maintain for less 48 h at room temperature
(Chew et al., 2019). The time will be even shorter at 37°C, at
which exposed functional substances (proteins and RNA) will be
rapidly degraded and metabolized. In fact, stability is an
important but often overlooked point in the research of
biomaterials loading exosomes, which should be given
sufficient attention in the further. Hydrogel encapsulated
exosomes was reported to protect them without degradation
and supply therapeutic effects with persistent exosomes delivery
(Riau et al., 2019). Li et al. (2021b) used the gelatin and laponite
to prepare nanocomposite hydrogels as a carrier for exosomes to
extend the time of BMSC-exosomes in the periodontal pocket
and enhance their osteoinductive function.
4.2 Enhancing local concentrations of
exosomes
The therapeutic effect of exosomes depends strongly on the
local concentrations. However, it is demanding to produce
exosomes in large quantities with high quality and purity,
making clinical applications of exosomes more expensive.
Additionally, free exosomes diffused out from the defect
rapidly, resulting in no exertion of exosomal cargo activity
(Riau et al., 2019). According to the research of Lai et al.
(2012), biodistribution proceeds in the stages of liver and
lungs for 30 min after direct injection of exosomes, and
exosomes are removed within 1 h–6 h after administration via
liver and kidney treatment. Thus, much research was performed
with the aim of enhancing the retention and sustained-releasing
of exosomes at the defect site. Xing et al. (2021) synthesized an
acellular extracellular matrix hydrogel coupled with adipose-
derived mesenchymal stem cell exosomes to regulate the
intervertebral disc microenvironment for the treatment of
intervertebral disc degeneration. The decomposition of the
hydrogel was slowed down, allowing exosomes to remain in
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the disc for up to 28 days Zhang Y. et al. (2021) fabricated an
injectable hyaluronic acid hydrogel encapsulated with umbilical
MSC-derived exosomes through three-dimensional (3D)
printing technology, and the hydrogel showed good sustained-
releasing features in the rat critical-size cranial defect model. Hu
et al. (2020) fabricated Gelatin methacrylate/nanoclay hydrogel
for sustained release of exosomes. The hydrogels with a 3D
matrix prevent the dispersion of exosomes and maintained
their local concentration, which enable the controlled release
of exosomes at bone defect sites (Holkar et al., 2021). In another
study, exosomes were incorporated into an injectable
thermosensitive hydrogel by constructing fusion peptides,
which also enhanced the retention of exosomes and improved
the biological activity of exosomes (Ma et al., 2022). Generally,
the consensus has been achieved that it is essential to enhance
local exosome concentrations at the bone defect site, and the
biomaterials loading exosomes should possess the property of
enhancing the retention and sustained-releasing of exosomes.
4.3 Enhancing exosomes activity
The hydrogel with 3D microenvironment can enhance exosome
activity and affect the interaction of integrin membrane protein
between cells and the cell matrix, which promotes cell proliferation
and differentiation in a bone regeneration environment. Yu W. et al.
(2022) encapsulated exosomes derived from periodontal ligament
stem cells into a hydrogel with 3D microenvironment, which
enhanced osteoinductive ability and significantly promoted bone
defect repair in rats. Another study demonstrated alginate hydrogels
combined with exosomes promoted osteogenesis by increasing cell-
exosomes interactions, cell aggregation, and long-term viability
(Holkar et al., 2021).
4.4 Optimizing the 3D distribution of
exosomes
Biomaterials with a highly porous and 3D structure mimic
the porosity, pore size, and interconnectedness of native bone
ideally. In bone defects repair, bioactive materials with good
mechanical properties not only provide temporary mechanical
support for the bone at the implant site, but also modulate
extracellular matrix formation, facilitate better cell-cell and
cell-matrix interactions, retain the cell morphology, provide
mechanical stimulations, and support cell growth and
exosomes secretion, the features which are akin to in vivo
systems (Tibbitt and Anseth, 2009). Matos et al. (2012)
showed that the lyophilized biomaterials created a more
homogenous interparticle spacing, allowed a more suitable
particle distribution and stabilization, then promoting a faster
bone regeneration with relevant clinical benefits. Similarly,
biocompatible 3D porous biomaterials ensured a uniform
spacing and stable distribution of MSC-exosomes compared
with compacted materials (Zha et al., 2021).
4.5 Antibacterial property
During bone defect healing, the bacterial infection is one of the
risk factors (Blair et al., 2015). Therefore, antibacterial property of
biomaterials should also be taken into consideration. Tan L. et al.
(2020) developed a food-grade probiotic-modified implant to
prevent methicillin-resistant Staphylococcus aureus infection and
accelerated bone integration. Liu et al. (2022) designed a
multifunctional hyaluronic acid (HA) hydrogel with antibacterial
property. Then, this group loaded plasma exosomes to this hydrogel
for promoting infected fracture healing (Yu C. et al., 2022). Mi et al.
(2022) combined engineered exosomes and a natural polymer HA
hydrogel, which performed an anti-inflammatory and antibacterial
function on fracture repair acceleration. As a cationic natural
polymer biomaterial, chitosan has anti-microbial property (Dai
et al., 2011), and many reports have shown cationic loaded
engineering exosomes could promote bone regeneration (Fan
et al., 2020;Shen et al., 2020;Wang et al., 2020;Wu et al.,
2021b;Bahar et al., 2022;Nikhil and Kumar, 2022), although
some of them did not look at the antibacterial property. The
biomaterials with antibacterial property have been designed in
some studies, but these materials are still rarely used for bone
defect repair, which merits further investigation.
4.6 Adapting to irregular bone defects
Clinical bone defects caused by trauma, neoplasia,
infection or corrective osteotomies are always irregular.
Hence, biomaterials should have the injectable property to
fill irregular defects and promote in situ bone tissue
regeneration. Xing et al. (2021) constructed an injectable
thermosensitive hydrogel system via a coordinative crossing
of ADSC-derived exosomes and acellular extracellular matrix
hydrogels to effectively protect nucleus pulposus cells from
pyroptosis after intervertebral disc degeneration. By taking
advantage of injectable, reversible, and thermosensitive
abilities, Tao et al. (2021) used PDLLA-PEG-PDLLA
triblock copolymer gels as a carrier of synovium
mesenchymal stem cells-derived exosomes for intra-
articular injection to prevent osteoarthritis progression.
Additionally, multiple biomaterials, including chitosan
hydrogel (Fan et al., 2020;Wu et al., 2021b),
nanocomposite hydrogel (based on gelatin and Laponite)
(Liu et al., 2021b), PG/TCP (PEGMC with β-TCP) (Zhang
B. et al., 2021), HA hydrogel (Yu C. et al., 2022), alginate
(Huang et al., 2020;Holkar et al., 2021), Gel-ADH (hydrazide
grafted gelatin) (Lin et al., 2022), silk fibroin (Shen et al.,
2022), hyaluronic acid (Yang S. et al., 2020;Sang et al., 2022)
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and SIS-CA (small intestinal submucosa (SIS) with propionic
acid (CA)) hydrogel (Ma et al., 2022)werereportedtoadapt
irregular bone defects and were used to carry exosomes for
skeletal regeneration. Generally, although local injection
therapy is not suitable for certain types of bone
regeneration, such as spinal cord repair (Han M. et al.,
2022), injectable biomaterials loading engineering exosomes
have been used extensively to repair irregular bone defects.
4.7 Other properties
In a moist environment, the favorable adhesion of
biomaterials is essential for in situ bone regeneration (Hasani-
Sadrabadi et al., 2020;Li et al., 2020;Zhang FX. et al., 2021).
Inspired by mussel materials, which exhibit underwater robust
adhesion (Gao Z. et al., 2018), Zhang and the colleague (2021b)
prepared a hydrogel with high bonding strength to the wet
surface using a crosslinked network of alginate-dopamine,
chondroitin sulfate, and regenerated silk fibroin, which
promote cartilage defect regeneration by combining with
BMSCs-exosomes. Peptide-modification is another strategy for
enhancing biomaterial adhesion. Li et al. (2020) prepared a
biomaterial with high adhesion by modifying HA hydrogel
with the PPFLMLLKGSTR peptide, which could locally deliver
human placenta amniotic membrane mesenchymal stem cell-
derived exosomes in spinal cord tissue.
Biomaterials with thermo-sensitivity are also of particular
concern for their property, changing between a liquid state and a
solid-state based on the ambient temperature (López-Noriega et al.,
2014;Ni et al., 2014). Several thermo-sensitive biomaterials have
been used in bone defect repair (Fu et al., 2012;Kim et al., 2018;Yu
et al., 2020;Wang QS. et al., 2021). Further, Ma et al. (2022) designed
a novel thermo-sensitive biomaterial by loading BMSCs-exosomes
with SIS-CA hydrogel to regulate bone regeneration.
Collectively, the bio-functional materials not only provide a
scaffold or carrier for engineering exosomes, but also play an
essential role by their own variety properties. Along with major
advancements in chemical engineering techniques, more and
more novel biomaterials with various properties have been
synthesized for bone regeneration. In the future, selection of
appropriate biomaterials to integrate engineering exosomes
should be one of the leading focuses of bone defect repair.
5 Conclusion and perspective
Coexistence of challenges and opportunities have greatly
stimulated the study of engineering exosomes for bone
regeneration in the past 10 years. In the present review, we
mainly addressed the molecular basis of exosomal cargos, the
strategies of engineering exosomesandthepropertiesofexosome-
integrated biomaterials required for bone regeneration. The research
about engineering exosomes for bone defect repair is undeniably in
its infancy. The rapid development of engineering exosomes is
impeded by several key challenges, especially the consistency of
exosomes production. As a result, these difficulties inspired the
development of new and cutting-edge approaches, often distinct
from those in the conventional study of cells, to address both
exosome production and function. Refining the isolation,
purification and storage techniques of exosomes may be an
effective means of improving the consistency of exosomes
production (Colao et al., 2018;Zeng et al., 2022). Additionally,
excellent biomaterials emerged continuously, which greatly
promoted research self-renewal. The effective combination of
engineering exosomes and biomaterials will be greater than the
sum of their parts and exhibit synergy effects in bone
regeneration. We optimistically foresee that novel biomaterials will
be constructed and more sophisticated engineering exosomes will be
implemented for bone tissue regeneration. This huge progress is sure
to benefit both biomedical research and therapeutic modalities in the
field of bone regeneration.
Author contributions
Conceptualization, SM, YL, and DP; writing—original draft
preparation, SM, YZ, and SL; writing—review and editing, SM,
AL, and DP; visualization, SM and YZ; funding acquisition, DP,
YL, and SM. All authors have read and agreed to the published
version of the manuscript.
Funding
This work was supported by National Natural Science
Foundation of China (No. 81870798 and 82170927) and the
Fundamental Research Funds for the Central Universities, Xi’an
Jiaotong University (xzy012021066).
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Frontiers in Bioengineering and Biotechnology frontiersin.org11
Ma et al. 10.3389/fbioe.2022.1091360

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