Scaffold-Mediated Immunoengineering as Innovative Strategy for Tendon Regeneration

Abstract

Tendon injuries are at the frontier of innovative approaches to public health concerns and sectoral policy objectives. Indeed, these injuries remain difficult to manage due to tendon’s poor healing ability ascribable to a hypo-cellularity and low vascularity, leading to the formation of a fibrotic tissue affecting its functionality. Tissue engineering represents a promising solution for the regeneration of damaged tendons with the aim to stimulate tissue regeneration or to produce functional implantable biomaterials. However, any technological advancement must take into consideration the role of the immune system in tissue regeneration and the potential of biomaterial scaffolds to control the immune signaling, creating a pro-regenerative environment. In this context, immunoengineering has emerged as a new discipline, developing innovative strategies for tendon injuries. It aims at designing scaffolds, in combination with engineered bioactive molecules and/or stem cells, able to modulate the interaction between the transplanted biomaterial-scaffold and the host tissue allowing a pro-regenerative immune response, therefore hindering fibrosis occurrence at the injury site and guiding tendon regeneration. Thus, this review is aimed at giving an overview on the role exerted from different tissue engineering actors in leading immunoregeneration by crosstalking with stem and immune cells to generate new paradigms in designing regenerative medicine approaches for tendon injuries.

Full text

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Citation: Russo, V.; El Khatib, M.;
Prencipe, G.; Varona, A.C.; Citeroni,
M.R.; Mauro, A.; Berardinelli, P.;
Faydaver, M.; Haidar Montes, A.A.;
Turriani, M.; et al. Scaffold-Mediated
Immunoengineering as Innovative
Strategy for Tendon Regeneration.
Cells 2022,11, 266. https://doi.org/
10.3390/cells11020266
Academic Editors: Alberto Sensini,
Gordon Blunn, Martijn van
Griensven and Lorenzo Moroni
Received: 6 December 2021
Accepted: 10 January 2022
Published: 13 January 2022
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Copyright: © 2022 by the authors.
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cells
Review
Scaffold-Mediated Immunoengineering as Innovative Strategy
for Tendon Regeneration
Valentina Russo 1, † , Mohammad El Khatib 1, † , Giuseppe Prencipe 1, * , Adrián CerveróVarona 1,
Maria Rita Citeroni 1, Annunziata Mauro 1, Paolo Berardinelli 1, Melisa Faydaver 1,
Arlette A. Haidar Montes 1, Maura Turriani 1, Oriana Di Giacinto 1, Marcello Raspa 2, Ferdinando Scavizzi 2,
Fabrizio Bonaventura 2, Liliana Liverani 3, Aldo R. Boccaccini 3and Barbara Barboni 1
1Unit of Basic and Applied Sciences, Faculty of Biosciences and Agro-Food and Environmental Technologies,
University of Teramo, 64100 Teramo, Italy; vrusso@unite.it (V.R.); melkhatib@unite.it (M.E.K.);
acerverovarona@unite.it (A.C.V.); mrciteroni@unite.it (M.R.C.); amauro@unite.it (A.M.);
pberardinelli@unite.it (P.B.); mfaydaver@unite.it (M.F.); aahaidarmontes@unite.it (A.A.H.M.);
mturriani@unite.it (M.T.); odigiacinto@unite.it (O.D.G.); bbarboni@unite.it (B.B.)
2Institute of Biochemistry and Cellular Biology (IBBC), Council of National Research (CNR),
Campus International Development (EMMA-INFRAFRONTIER-IMPC), 00015 Monterotondo Scalo, Italy;
mraspa@emma.cnr.it (M.R.); fscavizzi@emma.cnr.it (F.S.); bonaventura@emma.cnr.it (F.B.)
3Department of Materials Science and Engineering, Institute of Biomaterials,
University of Erlangen-Nuremberg, 91058 Erlangen, Germany; lialiana.liverani@fau.de (L.L.);
aldo.boccaccini@fau.de (A.R.B.)
*Correspondence: gprencipe@unite.it
† These authors share first authorship.
Abstract:
Tendon injuries are at the frontier of innovative approaches to public health concerns
and sectoral policy objectives. Indeed, these injuries remain difficult to manage due to tendon’s
poor healing ability ascribable to a hypo-cellularity and low vascularity, leading to the formation
of a fibrotic tissue affecting its functionality. Tissue engineering represents a promising solution for
the regeneration of damaged tendons with the aim to stimulate tissue regeneration or to produce
functional implantable biomaterials. However, any technological advancement must take into
consideration the role of the immune system in tissue regeneration and the potential of biomaterial
scaffolds to control the immune signaling, creating a pro-regenerative environment. In this context,
immunoengineering has emerged as a new discipline, developing innovative strategies for tendon
injuries. It aims at designing scaffolds, in combination with engineered bioactive molecules and/or
stem cells, able to modulate the interaction between the transplanted biomaterial-scaffold and the
host tissue allowing a pro-regenerative immune response, therefore hindering fibrosis occurrence at
the injury site and guiding tendon regeneration. Thus, this review is aimed at giving an overview
on the role exerted from different tissue engineering actors in leading immunoregeneration by
crosstalking with stem and immune cells to generate new paradigms in designing regenerative
medicine approaches for tendon injuries.
Keywords:
tendon regeneration; tissue engineering; immunoengineering; scaffold; electrospinning;
immune response; stem cells; immune cells; biomolecules; mechanotransduction
1. Introduction
Tendinopathies are among the most difficult orthopedic injuries to be managed, which
in turn imply the development of advanced approaches to fulfill public health challenges
and sectoral policy objectives. They represent around 30% of musculoskeletal disorders
with multiple risk factors recognized, including overuse, aging, and metabolic diseases (i.e.,
obesity and diabetes) [
1
,
2
]. The associated socioeconomic burden is over EUR 180 billion
in the USA and EU, with a forecast of +25% over the next five years as a consequence of
the absence of an efficacious therapeutic solution and variations in life expectancy, lifestyle
Cells 2022,11, 266. https://doi.org/10.3390/cells11020266 https://www.mdpi.com/journal/cells

Cells 2022,11, 266 2 of 40
and working conditions [
3
]. In veterinary medicine around 46% of racehorses are affected
by tendinopathy that in turn reduces their performance, generating a negative economic
impact of EUR 400 billion worldwide [4].
Tendinopathies are normally characterized by reduced mobility associated with
chronic tendon pain and a consequent impairment in the movements that compromises
everyday life. In humans, the Achilles, rotator cuff, patellar and forearm flexor and extensor
tendons are the most vulnerable [
3
], while the superficial digital flexor tendon (SDFT) is
the most affected in athletic horses and Achilles tendons in dogs [
5
]. Unfortunately, sponta-
neous tendon healing is ineffective due to the tissue low cellularity and hypo-vascularity [
6
].
As a consequence, any tissue damage is only partially repaired with a disorganized extracel-
lular matrix (ECM) production/fibrotic tissue, with reduced biomechanical properties [
1
,
2
].
Indeed, inflammation in tendinopathy can persist through key inflammatory mediators
which contribute to disrupting the tissue ECM [
7
]. In detail, the increasing number of
inflammatory cells after tendon injury leads to an imbalance between pro-inflammatory
factors, provoking the degradation of ECM [
8
]. Infiltrating immune cells provoke inflamma-
tion due to the activation of inflammatory mediators’ pathways, comprising cytokines, e.g.,
tumor necrosis factor-
α
(TNF-
α
), interleukin-1
β
(IL-1
β
), IL-6, and prostaglandins including
prostaglandin E2 (PGE
2
), hence promoting pro-inflammatory macrophage (M1) and T cell
activity. The released inflammatory cytokines exhibit different roles, including the upregula-
tion of vascular endothelial growth factor (VEGF) synthesis together with the enhancement
of the production of metalloproteases (MMPs), such as MMP-1, MMP-3, MMP-8, MMP-9,
and MMP-13, implicated in inducing matrix destruction [
8
]. Tenocytes play an accountable
role in tissue remodeling and repair due to the presence of immune receptors on their cell
surface. Upon activation toward an inflammatory phenotype, tenocytes start secreting
inflammatory mediators in both autocrine or paracrine manner that can modulate the
inflammatory response and ECM remodeling after injury [
9
]. Thus, to progress in tendon
healing, it is crucial to turn off the inflammatory process and modulate the tendon immune-
sensing compartment [
10
,
11
]. These evidence on the modulation of inflammation in the
resolution of tendinopathies could represent at the same time a novel therapeutic strategy.
If inflammation would persist in damaged tendons, possible ulterior damage and degener-
ative states might lead to chronic tendon injuries which most of the time result in tendon
ruptures [
12
]. Over the last few decades, researchers have been developing new strategies
based on tissue engineering (TE) approaches as an alternative solution to the conventional
treatments for tendon ruptures [
13
]. That being said, to date there is no available thera-
peutic strategy able to restore the normal tendon functionality for force transmission and
body movements [
4
,
14
]. Given the anatomy and biomechanical function of the tendon, the
development of a tendon biomimetic scaffold, which mimics the collagen parallel structure
and the biomechanics of native ECM of tendon tissue, could be game-changing for tendon
regeneration [
15
–
19
]. Scaffolds used in tendon TE are considered as temporarily ECM
replacement whose aim is guiding the neo-formation and the deposition of ECM while
parallelly degrading throughout the regeneration process [
20
]. These could be addressed
through two complementary pathways including the activation of endogenous and/or
exogenous cell regeneration mechanisms that could be driven by the functionalization of
the scaffold with stem and/or progenitor cells [
21
]. Therefore, scaffolds are frequently
utilized in combination with growth factors and stem cells whose aim is to support the
tissue healing both mechanically and biologically. Engineering functional tendon scaffolds
will thus require new approaches for the design of scaffolds with multi-scale resolution and
biomimetic features in terms of biophysical and biological properties, as well as cellular
regeneration to control cell spatial distribution and/or signaling molecules for modulating
cellular fate. Only a highly integrated approach that can combine under a single system
the multiple biophysical, biochemical, and biological cues controlling tenogenesis and
inflammation will allow the fabrication of functional tendon biomimetic scaffolds that can
be transplanted
in vivo
. Recent studies have demonstrated that modulating the intrinsic
(fiber alignment and diameter size, pore size, surface modification, incorporation of bioac-

Cells 2022,11, 266 3 of 40
tive molecules) and extrinsic (scaffold degradation, oxygen concentration and mechanical
stimuli) features of the designed scaffolds [
22
] activates an inflammatory cascade at the
injury site, which in turn might negatively or positively affect tendon regeneration [
23
,
24
].
In this context, immunoengineering has emerged as new discipline, which involves dif-
ferent strategies that enhance the interaction between the transplanted biomaterial-scaffold
and the host tissue, hence modulating the immune response and hindering fibrosis occur-
rence at the injury site [
25
]. An ideal scaffold must change the tendon pro-inflammatory
response and enhance its regeneration. Understanding the effect of scaffolds on modulating
the immune response and promote tendon regeneration is fundamental, as it allows to
fabricate “immunoinformed” scaffolds that incorporate specific design characteristics to
actively modulate the host tissue immune response and are able to co-adjuvate the switch
from a pro-inflammatory response towards a pro-regenerative one [
26
]. Indeed, when
implanting a biomaterial scaffold, the immune system is the first responder to the foreign
body [
22
]. The behavior of immune cells, that have large and diverse secretomes, can be
tuned by the intrinsic and extrinsic features of the biomaterial scaffold [
22
,
25
]. Targeting
these early responders to the scaffold can influence the local microenvironment and attract
key contributors in the subsequent regeneration process, such as stem cells and vascular-
ization [
27
,
28
]. This less explored feature in TE refers to applying immunoengineering
biomaterial strategies, in which optimally designed and functionalized scaffolds allow to
modulate host inflammatory response and stimulate tissue regeneration [
27
,
29
–
31
] or even
potentiate stem cells’ immunomodulatory functions [32,33].
The relevance of the topic can be deduced by analyzing the Scopus database and per-
forming a scientometric approach on the available literature using “immunomodulation”,
“immunomodulatory”, “immunoregenerative”, “immunoregeneration”, and “immuno-
engineering” as keywords. The analysis revealed that 16,352 out of 124,997 (13%) belong
to the musculoskeletal tissues (Figure 1A). A deep detailed database research allowed
to determine that bones, muscles, and cartilages cover 67%, 26%, and 5%, respectively,
whereas the remaining percentage (2%) belong to tendons and ligaments (Figure 1B).
Cells 2022, 11, x FOR PEER REVIEW 3 of 41
Recent studies have demonstrated that modulating the intrinsic (fiber alignment and di-
ameter size, pore size, surface modification, incorporation of bioactive molecules) and ex-
trinsic (scaffold degradation, oxygen concentration and mechanical stimuli) features of
the designed scaffolds [22] activates an inflammatory cascade at the injury site, which in
turn might negatively or positively affect tendon regeneration [23,24].
In this context, immunoengineering has emerged as new discipline, which involves
different strategies that enhance the interaction between the transplanted biomaterial-
scaffold and the host tissue, hence modulating the immune response and hindering fibro-
sis occurrence at the injury site [25]. An ideal scaffold must change the tendon pro-inflam-
matory response and enhance its regeneration. Understanding the effect of scaffolds on
modulating the immune response and promote tendon regeneration is fundamental, as it
allows to fabricate “immunoinformed” scaffolds that incorporate specific design charac-
teristics to actively modulate the host tissue immune response and are able to co-adjuvate
the switch from a pro-inflammatory response towards a pro-regenerative one [26]. In-
deed, when implanting a biomaterial scaffold, the immune system is the first responder
to the foreign body [22]. The behavior of immune cells, that have large and diverse secre-
tomes, can be tuned by the intrinsic and extrinsic features of the biomaterial scaffold
[22,25]. Targeting these early responders to the scaffold can influence the local microenvi-
ronment and attract key contributors in the subsequent regeneration process, such as stem
cells and vascularization [27,28]. This less explored feature in TE refers to applying im-
munoengineering biomaterial strategies, in which optimally designed and functionalized
scaffolds allow to modulate host inflammatory response and stimulate tissue regeneration
[27,29–31] or even potentiate stem cells’ immunomodulatory functions [32,33].
The relevance of the topic can be deduced by analyzing the Scopus database and
performing a scientometric approach on the available literature using “immunomodula-
tion”, “immunomodulatory”, “immunoregenerative”, “immunoregeneration”, and “im-
munoengineering” as keywords. The analysis revealed that 16,352 out of 124,997 (13%)
belong to the musculoskeletal tissues (Figure 1A). A deep detailed database research al-
lowed to determine that bones, muscles, and cartilages cover 67%, 26%, and 5%, respec-
tively, whereas the remaining percentage (2%) belong to tendons and ligaments (Figure
1B).
Figure 1. The comparative scientometric analysis of available publications on the Scopus database
by using the terms “immunomodulation”, “immunomodulatory”, “immunoregenerative”, “immu-
noregeneration”, and “immunoengineering” reveals that: (A) only 13% of the total publications re-
fer to the musculoskeletal tissues. (B) A deep analysis concerning the musculoskeletal tissues
demonstrated that bones are the most studied tissue in this field (67%) followed by muscles (26%),
cartilages (5%), and finally with the least publications number for tendons and ligaments with 1%
each.
Figure 1.
The comparative scientometric analysis of available publications on the Scopus database by
using the terms “immunomodulation”, “immunomodulatory”, “immunoregenerative”, “immunore-
generation”, and “immunoengineering” reveals that: (
A
) only 13% of the total publications refer to
the musculoskeletal tissues. (
B
) A deep analysis concerning the musculoskeletal tissues demonstrated
that bones are the most studied tissue in this field (67%) followed by muscles (26%), cartilages (5%),
and finally with the least publications number for tendons and ligaments with 1% each.
A further in-depth analysis of manuscripts available on tendons and ligaments was
used to identify the main topics to design the present review in which a total of 304 articles
were found (Figure 2). In more detail, 197 articles (65%), belonging to the following key-

Cells 2022,11, 266 4 of 40
words “inflammatory”, “cytokines”, “interleukins”, “macrophage”, “immune response”,
“immune system”, and “immune cell” concern the immune response impact tendinopathy-
mediated inflammatory reaction. Moreover, 71 articles (23%) consider the stem cell role in
improving tendon healing and regeneration by hindering and modulating the inflammatory
response
in vivo
. The remaining 36 articles (12%), which represent the keywords “scaffold”,
“biomaterial”, “electrospinning”, “electrospun”, “aligned/alignment”, and “biomimetic
scaffold” focused on scaffolds and, in particular, on electrospinning made materials by
considering their effect in modulating the immune response in tendon ruptures. Hence, it is
of great importance to put light on the importance of tendon-like scaffolds and controlling
their characteristics in modulating the inflammatory response of the implanted scaffolds in
tendon in vivo applications.
Cells 2022, 11, x FOR PEER REVIEW 4 of 41
A further in-depth analysis of manuscripts available on tendons and ligaments was
used to identify the main topics to design the present review in which a total of 304 articles
were found (Figure 2). In more detail, 197 articles (65%), belonging to the following key-
words “inflammatory”, “cytokines”, “interleukins”, “macrophage”, “immune response”,
“immune system”, and “immune cell” concern the immune response impact tendinopa-
thy-mediated inflammatory reaction. Moreover, 71 articles (23%) consider the stem cell
role in improving tendon healing and regeneration by hindering and modulating the in-
flammatory response in vivo. The remaining 36 articles (12%), which represent the key-
words “scaffold”, “biomaterial”, “electrospinning”, “electrospun”, “aligned/alignment”,
and “biomimetic scaffold” focused on scaffolds and, in particular, on electrospinning
made materials by considering their effect in modulating the immune response in tendon
ruptures. Hence, it is of great importance to put light on the importance of tendon-like
scaffolds and controlling their characteristics in modulating the inflammatory response of
the implanted scaffolds in tendon in vivo applications.
Figure 2. The scientometric analysis conducted on Scopus database (304 total articles) with the aim
to assess the different research topics concerning the immunoregeneration of tendon discussed in
this review. The legend indicates the different keywords used in the research whereas the number
of total publications for each keyword is written inside the box.
Starting from these premises, the present review is designed to give an overview
about the scaffolds’ immunoengineering strategies for tendon TE applications, with focus
on electrospun scaffolds, applied to modulate the immunomodulatory properties of the
different immune and stem cells in vitro and in vivo. Moreover, the different molecular
pathways regulating the scaffold mediated immunomodulation are detailed and dis-
cussed.
2. Biomimetic Scaffolds Applied for Tendon TE
When designing a scaffold for tendon TE applications, many characteristics need to
be fulfilled, in particular concerning scaffold surface morphology and mechanical prop-
erties to mimic the native tendon tissue properties and fibrous structure. The tendon ECM,
which constitutes around 80% of the tendon, contains predominantly collagen, which rep-
resents 60% to 85% of the dry mass, while the remaining approximate 20% represents the
resident cells including tenocytes, tenoblasts, tenocyte progenitor/stem cells (TPSCs), and
endothelial cells [34,35]. Collagen molecules within the tendon are arranged hierarchi-
cally, and intercalated with a less fibrous, highly hydrated matrix, traditionally referred
to as the ground substance [36]. Collagen type I (COL1) is the most abundant collagen
Figure 2.
The scientometric analysis conducted on Scopus database (304 total articles) with the aim
to assess the different research topics concerning the immunoregeneration of tendon discussed in this
review. The legend indicates the different keywords used in the research whereas the number of total
publications for each keyword is written inside the box.
Starting from these premises, the present review is designed to give an overview
about the scaffolds’ immunoengineering strategies for tendon TE applications, with focus
on electrospun scaffolds, applied to modulate the immunomodulatory properties of the
different immune and stem cells
in vitro
and
in vivo
. Moreover, the different molecular
pathways regulating the scaffold mediated immunomodulation are detailed and discussed.
2. Biomimetic Scaffolds Applied for Tendon TE
When designing a scaffold for tendon TE applications, many characteristics need to be
fulfilled, in particular concerning scaffold surface morphology and mechanical properties
to mimic the native tendon tissue properties and fibrous structure. The tendon ECM,
which constitutes around 80% of the tendon, contains predominantly collagen, which
represents 60% to 85% of the dry mass, while the remaining approximate 20% represents the
resident cells including tenocytes, tenoblasts, tenocyte progenitor/stem cells (TPSCs), and
endothelial cells [
34
,
35
]. Collagen molecules within the tendon are arranged hierarchically,
and intercalated with a less fibrous, highly hydrated matrix, traditionally referred to as the
ground substance [
36
]. Collagen type I (COL1) is the most abundant collagen molecule
and is responsible for the fibrous structure [
37
,
38
], followed by collagen type III (COL3)
which is normally restricted to the endotenon and sheets. In case of spontaneous tendon
healing, instead, COL3 is abundantly found representing the first collagen to be produced
in high quantity [
38
,
39
]. In this phase, COL3 fibers are randomly arranged forming a sort

Cells 2022,11, 266 5 of 40
of scaffold for the repairing site, accompanied with an increased cellularity. When the
remodeling stage occurs during the tendon healing, there is a decrease of cellularity and
matrix production. At this stage, COL3 is replaced by COL1 although with a favorable
higher ratio of COL3. This condition induces a reduced strength of the repaired tissue. As
a consequence, the tendon thickens and has a lower mechanical strength; thus, the tendon
quality and its functional activity are inferior to that of a healthy tendon [38,39].
Healthy tendons are characterized by their elasticity, good flexibility, and high me-
chanical strength. Indeed, the materials must have appropriate mechanical properties,
comparable to tissue target of the regeneration and sufficient to maintain the morphology
of the biomaterial during tissue development [34,40].
The tendon biomimetic scaffold must be designed with teno-inductive characteristics
to induce recruited progenitor cells or use undifferentiated stem cells for their differen-
tiation towards the tenogenic lineage [
16
,
17
,
40
,
41
]. Moreover, the designed tendon-like
scaffold should be teno-conductive to promote tendon growth and the surrounding tendon
ingrowth, thus directing neo-tendon deposition [
17
,
19
,
41
–
43
]. The tendon-like scaffold to
be implanted should be integrated into the surrounding host tendon. The teno-integration
property depends on teno-induction and teno-conduction. It is referred to the direct
anchorage of tendon within the damaged area without the formation of a scar fibrotic
tissue [41].
A key important aspect to be considered when designing tendon-like scaffold is the im-
munomodulation [
22
]. Two concepts should be taken into consideration: immunotolerance
and immunoinduction. The fabricated tendon-like scaffold should be immunotolerated, so
that it should not induce an inflammatory response within the implanted site by reducing
the activation of immune cells after transplantation. Additionally, the scaffold should be
immune-inductive, thus designed to modulate a favorable immune response by regulating
the intracellular and cell surface receptors presented on the host immune cells, such as
toll-like receptors (TLRs), dendritic cells (DCs), antigen-specific T-cell receptor (TCR), and
B-cell receptor (BCR), as well as by supporting immunomodulation, for example by shifting
the pro-inflammatory M1 macrophages into anti-inflammatory M2 [
24
,
31
,
44
,
45
], which is
discussed in detail in the next chapter.
Although different techniques have been used to fabricate scaffolds that aim to replace
damaged tendons including sponges [
43
,
44
], freeze-drying [
45
–
48
], extrusion [
49
], and
electrochemically aligned collagen [
50
–
52
], electrospinning is considered one of the most
effective technique due to its versatility and applicability, as well as its ability to produce
fibrous matrices that resemble native tendon architecture, with the possibility to control the
fiber orientation and alignment [17–19,40,53–57].
The achieved fibrous structure should mimic the hierarchical morphology of the native
tendon tissue characterized by collagen fascicles, fibers, and fibrils [
58
–
60
], confirming the
well-known biomimicry characteristics of the electrospun fibers relevant to support cell
adhesion, proliferation and differentiation [42,50,61–65].
The understanding of the relationship between scaffold properties and cell differentia-
tion towards tenogenic lineage is relevant because the scaffold properties can be tailored
by optimizing the electrospinning process parameters. The fiber orientation is a key point
for the obtainment of a scaffold suitable for tendon TE and it is usually adjusted by an
appropriate selection of the fiber collector type and configuration. Typical electrospun
fiber diameters can range from 10 nm to 10
µ
m [
66
]. In tendon TE, the electrospun fibrous
scaffolds should be in the micrometer range since microfibers mimic the physiological
architecture of collagen fibers where tenocytes reside [
18
,
62
]. Indeed, it has been demon-
strated that electrospun microfibers allow higher cell alignment and teno-differentiation
accompanied with an improvement in the alignment of ECM, avoiding the formation of
scar fibrotic tissue and promoting tissue healing [
62
,
67
]. Instead, electrospun nanofibers,
which mimic collagen fibril size, have shown to stimulate the proliferative phase of tendon
repair [62,67].

Cells 2022,11, 266 6 of 40
The electrospinning technique allows the fabrication of scaffolds, which possess different
biomimetic tendon-like shapes adequate to be used for tendon TE
applications [17–19,54,68–72]
.
Indeed, bundles and yarn scaffolds are other types of tendon-like scaffolds characterized as
filament and twisted filaments of electrospun aligned fibers, respectively, which better mimic
the hierarchical tendon architecture, as for example tendon fascicle crosslinked electrospun
collagen nanofibers [
56
,
57
]. Multilayer scaffolds [
73
], stacked and braided scaffolds, represent
scaffolds used in tendon TE obtained by assembling meshes of electrospun aligned fibers using
crosslinking approaches to improve their mechanical properties [42,74,75].
3. Immune Response Induced by Scaffold Implantation
The implantation of scaffold
in vivo
starts a cascade of reaction called foreign body
response (FBR), evolved by the host tissue that usually lasts for 1 or 2 weeks (Figure 3). FBR
can either determine the failure of the implanted scaffold or progress in tissue regeneration
mainly induced by the shift from pro-inflammatory M1 macrophages to anti-inflammatory
M2 macrophages and T helper cells (i.e., Th2) [
32
,
38
]. In this field, researchers are working
on developing new strategies that aim at suppressing and harnessing the immune system to
promote scaffold tolerance. Immunotolerance represents the functional unresponsiveness
of the immune system towards cells and tissues [
76
]. Scaffolds should avoid the risk of
inducing an aberrant inflammatory response when being implanted
in vivo
. A lack of an
immunomodulatory response in terms of macrophage M2 and Th2 results in frustrated
phagocytosis, which is detrimental to tissue repair [32].
In this process, innate and adaptive immune response are both in charge [
32
]. In detail,
when a scaffold is implanted in a host tissue, blood-related proteins are adsorbed on the
surface of implanted scaffold which activate the coagulation process allowing the formation
of a temporary matrix characterized by an initial adsorption of albumin, substituted then by
globulins that are in turn replaced by fibrinogen, fibronectin, factor XII and high molecular
weight kininogens (Figure 3A) [
77
]. Fibrinogen accumulation represents a key role in FBR;
its spontaneous adsorption appears to initiate the acute inflammatory response. Innate
immune cells, including neutrophils, mast cells, and monocytes/macrophages are then
recruited and accumulate in the region between the scaffold implants and the surrounding
tissue (Figure 3B) [78]. The recruited cells start to secrete pro-inflammatory cytokines and
chemokines, hence increasing the immune cell recruitment and promoting inflammation
(Figure 3C) [
32
]. Afterwards, immune cells from the adaptive immune response including
B cells, CD4+ and CD8+ T cells, natural killer (NK) cells, and innate lymphoid cells start to
release cytokines and chemokines within the implanted site [
32
,
79
–
81
]. The occurrence of
acute and chronic inflammation is followed by the formation of neovascularized connective
tissue, called granulation tissue. The permanent implant could contribute to chronic
inflammation documented by the presence of macrophages, lymphocytes, and foreign
body giant cells (FBGC) (Figure 3D). These last, resulted from the fusion of adherent
macrophages, are associated with the switch from pro-inflammatory M1 to pro-regenerative
M2 phenotypes and from T helper 1 (Th1) cells to Th2, that could attempt to increase their
regenerative functionality (Figure 3E) [
24
]. Both macrophage phenotypes are transient,
which means that polarized macrophages will re-polarize to a different phenotype based on
environmental needs. A previous work showed that the recruitment of pro-regenerative M2
macrophages promotes tendon regeneration [
82
]. Thus, in order to reach an optimal tissue
regeneration, there is a need to tune the type and timing of the inflammatory components.
In this context, while much emphasis has been placed by researchers on varying
electrospun scaffold properties to improve target cell adhesion, infiltration, integration
and their teno-differentiative responses [
34
,
40
], it is crucial to focus the attention on the
interaction between immune cells and a specific scaffold design [
30
,
78
]. Thus, under-
standing how to modulate scaffold microenvironmental cues to monitor the immune cell
response, and in particular M2:M1 ratio, is crucial in the development of next-generation
immunomodulated scaffolds able to positively promote tissue remodeling, incorporation,
and regeneration [
26
,
30
]. The activation state of the inflammatory response can be fur-

Cells 2022,11, 266 7 of 40
ther evaluated by analyzing the expression profile of released cytokines by the immune
cells [33,83–85].
Cells 2022, 11, x FOR PEER REVIEW 7 of 41
Figure 3. Interaction between scaffold and host tissue after implantation. The host starts a cascade of
reaction called foreign body response (FBR). (A) Blood-related proteins and circulated platelets are re-
cruited at the implantation site and are adsorbed on the surface of the scaffold allowing the activation of
the coagulation process. (B) Immune cells are recruited at the implantation site and are accumulated
between the scaffold and the surrounding tissue. (C) The immune cells start to secrete pro-inflammatory
cytokines and promote inflammation. (D) The protracted presence of the scaffold and the persistent in-
flammation accompanied with the increased number of M1 pro-inflammatory cytokines leads to a con-
tinuous activation of tenocytes which secrete more collagen, which contributes to the formation of fi-
brous capsule and the rejection of the scaffold. (E) The switch of macrophages towards the anti-inflam-
matory/pro-regenerative phenotype M2 promotes tendon regeneration and facilitates the healing pro-
cess.
In this context, while much emphasis has been placed by researchers on varying elec-
trospun scaffold properties to improve target cell adhesion, infiltration, integration and
their teno-differentiative responses [34,40], it is crucial to focus the attention on the
Figure 3.
Interaction between scaffold and host tissue after implantation. The host starts a cascade
of reaction called foreign body response (FBR). (
A
) Blood-related proteins and circulated platelets
are recruited at the implantation site and are adsorbed on the surface of the scaffold allowing the
activation of the coagulation process. (
B
) Immune cells are recruited at the implantation site and are
accumulated between the scaffold and the surrounding tissue. (C) The immune cells start to secrete
pro-inflammatory cytokines and promote inflammation. (
D)
The protracted presence of the scaffold
and the persistent inflammation accompanied with the increased number of M1 pro-inflammatory
cytokines leads to a continuous activation of tenocytes which secrete more collagen, which contributes
to the formation of fibrous capsule and the rejection of the scaffold. (
E
) The switch of macrophages
towards the anti-inflammatory/pro-regenerative phenotype M2 promotes tendon regeneration and
facilitates the healing process.

Cells 2022,11, 266 8 of 40
On the premise that tissue regeneration is intrinsically linked to the host immune
response, the immunoregenerative process could represent a key to regenerative medicine
strategies. Scaffolds must not only be passive supports for stem cells’ activity after im-
plantation to boost tissue regeneration. Indeed, scaffolds can be designed in a variety of
ways to modulate the inflammatory response as well as to influence stem cells’ activity.
In a more advanced way, these biocompatible structures must be designed to modulate
the immune cells’ response and avoid the procrastination of the inflammatory condition
by inhibiting the secretion of inflammatory cytokines, formation of fibrous capsule and
chronic inflammation [
30
]. Then, to reach this target, it is crucial to investigate the immune
cell–biomaterial cross-interactions and the consequences on the host response. Tuning
the degradation rate of the tendon-like scaffolds is another factor to take into account
since it may affect tendon regeneration. While slow degradation rate is attributed to the
formation of FBGCs accompanied with chronic inflammation response and the formation
of fibrous tissue, scaffolds with faster degradation rate exhibit high cell infiltration within
the construct, improving tendon regeneration [86].
4. Scaffold Immunoengineering Strategies for Tendon TE Applications
Immunoengineering has emerged recently as new discipline whose aim is to generate
scaffolds with immune-modulatory properties to improve the interactions between the im-
planted scaffolds and the host immune systems to enhance the regenerative process. It aims
at applying the principles and approaches of engineering by developing immunomodu-
lated scaffolds that induce a favorable host immune response [
25
] and, consequently, tissue
regeneration. This discipline fits well with tendon resolution, as in fact, any technological
advancement in tendon TE has to take into consideration that tendon healing is associated
to the blunting of the tissue inflammatory state [
10
,
11
]. Indeed, the immune system plays
a central role in each tendinopathy stage and regulates the processes of tissue repair by
immune cells and the secreted cytokines [10].
Although historically many researchers have worked on developing biologically inert
implantable scaffolds, others still working on fabricating ideal bioactive scaffolds aim at
not only preventing interaction between scaffolds and the immune system, but also at
improving their biological effect in co-adjuvating a healing and regenerative process within
the implantable site [
25
]. Two layers of specificity are the key elements for the safeness and
effectiveness of the developed scaffold-based technology: the antigen specificity and the
immunomodulatory specificity. While the first one is addressed to ensure that tolerance is
limited to only the cells and the tissues to be protected, the second one aims at ensuring that,
for example, regulatory T cells, without the cytotoxic ones, are stimulated for protection [
25
].
Thus, the immunoengineering biomaterial strategies have the aim, by using optimal
scaffolds, to modulate host inflammatory response and consequently stimulate tissue
regeneration [
29
,
30
] or even potentiate stem cells’ immunomodulatory function [
17
,
31
,
32
].
In fact, stem cells, including mesenchymal stem cells (MSCs) or amniotic-derived stem
cells, as the amniotic epithelial stem cells (AECs), represent important cell sources that
have been widely used in tendon applications, in which it has been demonstrated an
improvement in the inflammatory response and an amelioration in the tissue regeneration
due to their immunomodulatory properties [
34
,
82
,
87
]. The regenerative role of stem cells
passes through several modulation mechanisms of the immune response through their
paracrine functions which mediate their therapeutic potentials [82,87,88].
The interplay of inherent electrospun scaffold properties with those arising from the
interactions with the local environment, because of biomaterial interaction, is very complex.
Different immunoinformed approaches have been studied and applied to produce immune-
instructive niches made up of electrospun scaffolds including adjustment of: (a) the intrinsic
properties of the scaffolds such as geometry, topography, porosity, pore size, substrate
stiffness, and polymer and surface chemistry; (b) the temporal properties by modulating
degradation rate of the scaffolds; and (c) the environment to which the scaffold is subjected
including mechanical stimuli and oxygen concentration (Figure 4).

Cells 2022,11, 266 9 of 40
Cells 2022, 11, x FOR PEER REVIEW 9 of 41
The interplay of inherent electrospun scaffold properties with those arising from the
interactions with the local environment, because of biomaterial interaction, is very com-
plex. Different immunoinformed approaches have been studied and applied to produce
immune-instructive niches made up of electrospun scaffolds including adjustment of: (a)
the intrinsic properties of the scaffolds such as geometry, topography, porosity, pore size,
substrate stiffness, and polymer and surface chemistry; (b) the temporal properties by
modulating degradation rate of the scaffolds; and (c) the environment to which the scaf-
fold is subjected including mechanical stimuli and oxygen concentration (Figure 4).
Figure 4. Immunoregenerative strategies applied in tendon TE to modulate the immune response
of immune and stem cells.
Based on these premises and considering the native tendon architecture, in the next
paragraphs, only electrospun scaffolds with aligned topography and characteristics that
mimic tendon ECM are considered and discussed in detail by exploring separately their
effect either on immune (Section 4.1) or stem cells (Section 4.2).
4.1. Immuno-Induction of Scaffold on Immune Cells
As described in the previous section, the inflammation and regeneration processes
evolve different types of immune cells [89]. However, macrophages tend to be the most
studied cell type due to their critical function in guiding tendon tissue regeneration and
avoiding its fibrosis. In this section, the effect of intrinsic and extrinsic properties of elec-
trospun scaffolds on immune cells is discussed with a particular attention on macro-
phages (Figure 5).
Figure 4.
Immunoregenerative strategies applied in tendon TE to modulate the immune response of
immune and stem cells.
Based on these premises and considering the native tendon architecture, in the next
paragraphs, only electrospun scaffolds with aligned topography and characteristics that
mimic tendon ECM are considered and discussed in detail by exploring separately their
effect either on immune (Section 4.1) or stem cells (Section 4.2).
4.1. Immuno-Induction of Scaffold on Immune Cells
As described in the previous section, the inflammation and regeneration processes
evolve different types of immune cells [
89
]. However, macrophages tend to be the most
studied cell type due to their critical function in guiding tendon tissue regeneration and
avoiding its fibrosis. In this section, the effect of intrinsic and extrinsic properties of electro-
spun scaffolds on immune cells is discussed with a particular attention on macrophages
(Figure 5).
4.1.1. Intrinsic Properties of the Scaffold on Immune Cells
Topography Effect on Immune Cells
Immunomodulation through topographical cues was recently discovered to be able to
override the effects of surface chemistry in certain materials, especially in the first 6–48 h
after initial contact. After implantation, topographical cues at the micro or nanoscale guide
macrophage responses such as adhesion, spreading, activation, migration, and polarization.
Indeed, macrophages, like many other cell types, can sense mechanical properties of their
environment [
30
]. Murine macrophages tend to be unable to detect nano-topographical
features smaller than 150 nm, while fibroblasts and endothelial cells can detect smaller
topographies and show less spreading as feature size increases from 55 nm to 200 nm [
30
].

Cells 2022,11, 266 10 of 40
Cells 2022, 11, x FOR PEER REVIEW 10 of 41
Figure 5. Effects of electrospun scaffolds-based immunoregenerative strategies on macrophage po-
larization and stem cell immunomodulation.
4.1.1. Intrinsic Properties of the Scaffold on Immune Cells
Topography Effect on Immune Cells
Immunomodulation through topographical cues was recently discovered to be able
to override the effects of surface chemistry in certain materials, especially in the first 6–48
h after initial contact. After implantation, topographical cues at the micro or nanoscale
guide macrophage responses such as adhesion, spreading, activation, migration, and po-
larization. Indeed, macrophages, like many other cell types, can sense mechanical prop-
erties of their environment [30]. Murine macrophages tend to be unable to detect nano-
topographical features smaller than 150 nm, while fibroblasts and endothelial cells can
detect smaller topographies and show less spreading as feature size increases from 55 nm
to 200 nm [30].
Surface topography properties such as fiber alignment and diameter have demon-
strated a significant impact on the severity of inflammatory responses [24] and on the po-
larization of macrophages from inflammatory (M1) to anti-inflammatory/pro-regenera-
tive (M2) and vice versa (Table 1). For example, Garg et al. cultured mouse bone marrow-
derived macrophages (BMMΦs) on polydioxanone (PDO) scaffolds with different fiber
diameter of 0.35, 2.20, and 2.80 μm, showing a positive correlation between increasing
fiber diameters and expression of M2 markers [90]. Analogously, Wang et al. described
an increase of RAW264.7 M2 macrophages phenotype when engineered on electrospun
Figure 5.
Effects of electrospun scaffolds-based immunoregenerative strategies on macrophage
polarization and stem cell immunomodulation.
Surface topography properties such as fiber alignment and diameter have demon-
strated a significant impact on the severity of inflammatory responses [
24
] and on the po-
larization of macrophages from inflammatory (M1) to anti-inflammatory/pro-regenerative
(M2) and vice versa (Table 1). For example, Garg et al. cultured mouse bone marrow-
derived macrophages (BMM
Φ
s) on polydioxanone (PDO) scaffolds with different fiber
diameter of 0.35, 2.20, and 2.80
µ
m, showing a positive correlation between increasing
fiber diameters and expression of M2 markers [
90
]. Analogously, Wang et al. described
an increase of RAW264.7 M2 macrophages phenotype when engineered on electrospun
polycaprolactone (PCL) scaffolds with fiber diameter size of 5.59
µ
m rather than 0.69
µ
m,
both
in vitro
and
in vivo
[
91
]. Instead, Saino et al. studied the effect of varying the topolog-
ical properties of electrospun poly(L-lacticide) (PLLA) scaffolds in terms of fiber diameter
and fiber alignment on the activation of macrophage RAW 264.7 and their secretion of
pro-inflammatory cytokines and chemokines. In that study, four different types of fibrous
PLLA scaffolds with different diameter sizes were identified as follows: aligned microfibers
(1.60
±
0.25
µ
m), aligned nanofibers (0.55
±
0.16
µ
m), random microfibers (1.53
±
0.32
µ
m),
and random nanofibers (0.61
±
0.18
µ
m), and PLLA film (0.2 mm thick flat surface) were en-
gineered with macrophages RAW 264-7 for 24 h and 7 days culture. In comparison to films
and microfibrous scaffolds, nanofibrous PLLA scaffolds reduced the inflammatory response.
Furthermore, histological analysis revealed that the PLLA film showed a higher number of
FBGCs than the micro and nanofibrous scaffolds. Thus, the findings of Saino et al. show
that the diameter of electrospun PLLA fibers influences
in vitro
macrophage activation and
pro-inflammatory molecule secretion [29].

Cells 2022,11, 266 11 of 40
Table 1. Overview of the influence of different electrospun scaffolds properties on immune cells.
Scaffold Properties Material Parameters Immune Response Reference
Diameter size
PDO Different fiber diameter size
(0.35, 2.20, and 2.80 µm) Increasing fiber diameter → ↑
M2 macrophages expression [90]
PCL Different fiber diameters
(0.69 and 5.59 µm)
Increased fiber diameter size
(5.59 µm) → ↑ M2
macrophages expression [91]
Alignment
PCL
Random and aligned fiber
orientation; scaffolds
unmodified or extended to
macro-scale thicknesses of
3 or 10 mm
Expanded scaffolds ↑
regenerative answer and
thinner collagen fibrous
capsule compared to
unexpanded nanofiber
scaffolds
Aligned fibers (expanded to
3 mm) fewest ↓number of
giant cells
[92]
PLLA
Five different types of
scaffolds:
aligned microfibers, aligned
nanofibers, random
microfibers, random
nanofibers, and on film
Nanofibrous PLLA scaffolds
↓inflammatory response than
films and microfibrous
scaffolds
PLLA film ↑number of
foreign body giant cells than
the micro and nanofibrous
scaffolds
[29]
PCL Random and aligned fiber
orientation
Random fibers ↑
pro-inflammatory response
compared to aligned fibers [93]
PCL Random and aligned fiber
orientation
Aligned fibers →least
amount of monocyte
adhesion with a thinner
fibrous capsule and more
fibroblasts infiltration
compared to randomly
oriented fibers
[94]
Pore size PDO Different pore size (0.96,
10.57, and 14.73 µm)
14.73 µm pore size →M2
macrophage polarization,
↑Arginase I and ↓iNOS [90]
Mechanical stimulus
PCL 7 and 12% cyclic uniaxial
strains (0.8 Hz)
7% mechanical strain → ↑
MCP-1, IL-6, IL-10, and
MMP-9 (M2 markers)
12% strain →M1
proinflammatory phenotype
[95]
CE-UPy-PCL Cyclic strains:
0%, 8% and 14% strain at
0.8 Hz
High strains addressed a
pro-inflammatory condition [96]
PCL Static culture (1% constant
strain) and dynamic loading
(7% cyclic strain at 1 Hz)
Dynamic loading → ↑ CCR7
(M1 marker) [93]
Surface modification
PLLA Lubricating layer of chitosan
collagen and alginate
hydrogel
↓Protein adsorption [97]
PLGA CTS layer coating
↓inflammatory cells
recruitment and FBGCs
formation [98]
PLLA Two layers of PLLA
membranes combined into a
single layer
↓Adhesion to the tissues [99]
PCL: polycaprolactone; CE-UPy-PCL: ureido-pyrimidinone (UPy)-modified Chain Extended Polycaprolactone; PLLA:
poly(L-lactide); PDO: polydioxanone; PLGA: poly(lactic-co-glycolic acid); CTS: chitosan; ↑: increase; ↓: decrease.
Immune responses have been shown to also be influenced by fibers’ orientation. For
example, Schoenenberger et al. investigated the different macrophages’ outcomes when
seeded on aligned or randomly oriented electrospun PCL nanofiber substrates. In all the
experiments, a disorganized biomaterial fiber topography was enough to facilitate a pro-
inflammatory response in macrophages, tendon fibroblasts, and tendon tissue compared
to aligned fibers [
93
]. Cao et al., compared the ability of electrospun PCL scaffolds with
random and aligned fiber orientation on the adhesion of monocytes. Compared to ran-
domly oriented fibers, aligned fibers showed a significant decrease in monocytes’ adhesion.
The obtained results were also confirmed
in vivo
, showing that after 4 weeks, aligned fiber

Cells 2022,11, 266 12 of 40
scaffolds were surrounded by a significantly thinner fibrous capsule. Moreover, the aligned
fibers had more fibroblast infiltration, whereas the randomly oriented fibers accumulated
cells on the surface [
94
]. In a similar way, as compared to surface-restricted geometries,
the three-dimensional (3D) microstructure decreases inflammatory cytokine activity [
30
].
Another study on the orientation of scaffold was conducted by Jiang et al., who developed
polymeric scaffolds by electrospinning PCL and then modifying the resulting fibers to give
them different shapes: random or aligned. These scaffolds were then either left unmodified
or expanded to macro-scale thicknesses of 3 or 10 mm, respectively (expanded in 1 M
NaBH
4
solution for 1 h at room temperature). Jiang et al., detected that expanded scaffolds
promoted a regenerative answer and led to a thinner collagen fibrous capsule compared to
unexpanded nanofiber scaffolds. Moreover, macrophages were able to penetrate scaffolds
with randomly aligned fibers with extended thicknesses of 3 or 10 mm after subcutaneous
implantation in rats. On the other hand, the scaffolds with aligned fibers that were ex-
panded to 3 mm have shown to greatly support macrophage penetration with the fewest
number of giant cells, which was attributed according to the authors to the gap distance
between the aligned fibers [92].
The development of electrospun aligned fibers can be critical for the success of tendon
tissue regeneration since they lead to a higher cell infiltration with a lower inflammatory
response. The mechanisms through which this topography feature acts are discussed in the
Section 4.
Influence of Porosity and Pore Size on Immune Cells
Porosity and pore size represent two critical scaffolds’ immunomodulatory factors that
could affect not only the infiltration of biological molecules, including proteins or oxygen,
but also the cellular behavior (Table 1). Size and frequency of the pores can be important
cues for immunomodulation [
100
]. Porous implants are more easily vascularized and
have less fibrous encapsulation than non-porous implants [
30
]. Garg et al., demonstrated
that increasing pore size of PDO electrospun scaffolds (14.73
µ
m) directs macrophages
polarization by boosting the M2 phenotype compared to other studied pore sizes (0.96 and
10.57
µ
m). This was suggested by the increased expression of Arginase I and down
expression of M1 marker iNOS [
90
]. Therefore, this experiment revealed a link between
increased pore size and a shift away from M1 macrophages toward M2 macrophages.
A main issue of the scaffold design is represented by the achievement of a suitable and
high scaffold porosity, to maintain suitable mechanical properties for the target application.
Tissue engineering constructs with increased porosity and an extended conformation can
encourage pro-regenerative environments by altering macrophage function, as shown
above. This improvement in scaffold structure, however, can have a negative impact on me-
chanical strength, and in the case of tissue-mimicking implants for structural components,
the scaffold’s mechanical strength must match that of the native tissue [
101
]. Additionally,
El Khatib et al. noticed that poly(lactic-co-glycolic acid) (PLGA) fiber diameter size is
directly linked to the pore size of the fibers: by increasing the diameter, the pore size of
the fibers increases as well [
18
]. This finding sounds very interesting since the study of El
Khatib et al., also reported that doubling the fiber diameter (2.5
µ
m instead of 1.27
µ
m),
and as a consequence the previously stated increase in the fibers’ pore size, resulted in
worse mechanical features [
18
]. Therefore, this study is in accordance with Andorko and
Jewell, and highlights that increased pore size can have a negative impact on scaffolds’
mechanical strength. The modulation of scaffolds’ porosity and pore size represents a good
strategy for immunomodulatory purposes; however, it is necessary to find a compromise
to obtain the best balance between the porosity of electrospun scaffolds and tendon-like
mechanical properties.
Effect of Surface Modification and Biomaterial Chemistry on Immune Cells
For years, scientists have been working on changing the surface chemistry of im-
plants to module the inflammatory response of immune cells. Indeed, surface chem-

Cells 2022,11, 266 13 of 40
istry/hydrophobicity has been long known to have a significant effect on the adsorption
and denaturation of blood serum proteins, as well as on subsequent cell responses. In
addition, surface roughness/smoothness influences wettability or hydrophilicity which
in turn affects protein adsorption and cell adhesion [
24
]. Roughness is a propriety of the
surface biomaterials that can be present on a microscale, microroughness, and that could
enhance cell adhesion [
100
]. This propriety seems to be a stimulus factor for both M1
and M2 macrophages [
100
]. Several studies have shown that films with rough surfaces
in the nanometer scale (150 nm to 4500 nm) can reduce fibroblast proliferation [
102
–
104
].
Smoother surfaces (roughness <200 nm) on the other hand have been shown to facilitate
proliferation [
98
,
99
,
101
]. Furthermore,
in vivo
experiments have shown that when the sur-
face roughness is greater than 500
µ
m, a thinner fibrous capsule forms [
105
]. The findings
of these studies could be extended to electrospun nanofibers, but unlike films, these have a
higher surface roughness.
In this field, friction plays a crucial role in tendon regeneration and adhesion formation.
The friction between tissue and scaffold can be reduced by using a lubricant surface on
the engineered biomaterial to reduce tissue adhesion (TA) formation. Failure to create
friction-free motion results in TA, which in turn compromises the full tendon regeneration.
Incorporating materials with low friction efficiency against the surrounding ECM, such as
5-fluorouracil and hyaluronic acid (HA), could lower protein absorption on the electrospun
fibrous matrix (EFM) and minimize friction surface, leading to less tissue adhesion [
85
]
(Table 1). For example, Deepthi et al. developed electrospun PLLA aligned nanofibers, mim-
icking the aligned tendon collagen fiber bundle, that were layered with a thin lubricating
layer of chitosan (CTS) collagen and alginate hydrogel, simulating the glycosaminoglycans
of sheath ECM for tendon regeneration. The study results showed a significantly reduced
protein adsorption at the scaffold surface without affecting
in vitro
cell infiltration and
attachment up to 7 days [
97
]. Protein adsorption experiments revealed that due to the lack
of protein binding sites in alginate, protein adsorption onto alginate coated membranes
was lower [
85
]. Moreover, Cheng et al., proposed a new method to improve the lubrication
of electrospun PCL membranes by grafting 2-methacryloyloxyethyl phosphorylcholine on
their surfaces [
106
]. Aside from the incorporation of different materials or surface chemical
modification, appropriate physical structure design for EFMs could also reduce friction
and promote tendon gliding [
85
]. Wang et al., for example, electrospun two layers of PLLA
membranes and then used a shearing force to combine them into a single layer [
99
]. During
the
in vivo
degradation process, such dual-layer scaffolds provided an artificial space to
facilitate tendon gliding, which was beneficial for suppressing peritendinous adhesion and
promoting tendon healing [85].
The biomaterial chemistry has a central role in the immune response; dependent on the
different biomaterial nature, the inflammatory events could be modulated. Chemical prop-
erties of the material can affect macrophage function due to differences in the type and con-
formation of adsorbed proteins or charged regions on macrophage membranes. Currently,
graphene is considered the most appealing material in nanotechnology [
107
]. Sub-cytotoxic
concentrations of graphene family nanomaterials are able to modulate macrophages by
stimulating the secretion of Th1/Th2 cytokines and chemokines most likely due to the
TLR-dependent activation of the TLR-mediated and NF-
κ
B signaling pathway [
108
]. Fur-
thermore, it has been determined that a sub-cytotoxic dose of pristine graphene modulates
macrophage morphology by modifying actin assembly and reducing their ability to ad-
here to the ECM [
108
]. Graphene oxide (GO), which represents the result of graphene
oxidation to increase hydrophilicity and preparation of composites [
109
], has been shown
to possess immunomodulatory properties, making them involved in the modulation of
immune cells, neutrophils, macrophages, dendritic cells [
110
]. For example, GO sheets
induce macrophage polarization to the M1 phenotype, enhancing the pro-inflammatory
response [
111
]. Macrophage polarization towards the M1 lineage has also been observed
with GO functionalized with amino groups, with an increase of secretion of CCL-5, IL-
6, and IL-1
β
[
112
]. Furthermore, Mukherjee et al. determined that human neutrophils

Cells 2022,11, 266 14 of 40
exposed to small (50–300 nm) and large (10–40
µ
m) sheets of GO produce neutrophil extra-
cellular traps (NETs) that contribute in pathogen defense by immobilization and killing of
bacteria [
113
]. These immunomodulatory properties together with the mechanical prop-
erties [
114
–
117
] of graphene and graphene-based materials have aroused a great interest
in its usage in TE. It was only during this decade that GO was first incorporated into
polymeric electrospun nanofibers [
118
]. Currently, several research teams are addressing
their study on the evaluation of composites of this material with electrospun polymer
scaffolds for tendon repair, for review [
109
]. Further studies are needed to investigate the
employment other immunomodulatory biomaterials in the electrospinning technique. For
example, the already mentioned CTS could represent a promising choice in TE scaffolds’
design [
119
]. However, CTS immunomodulatory effects have to be explored since its pro-
or anti-inflammatory role depends on the context [
119
]. Moreover, there are only few
reports about the electrospinning of pure CTS, and its usage in this field is limited due to
its high viscosity [120].
4.1.2. Effect of Scaffold Degradation on Immune Cells
The degradation rate of scaffolds can be influenced by the type of the polymer used.
Moreover, the scaffold degradation rate should ideally be synchronized with the new
healthy tissue formation, in fact the scaffold should provide sufficient mechanical and
dimensional support to the new tissue, while it degrades [
121
]. The main synthetic poly-
mers used for fabrication of electrospun scaffold for tendon TE applications belong to
the aliphatic polyester family. These polymers are biodegradable from which one of the
material’s basic component, lactic acid, is a cellular metabolite. These polyester-based
scaffolds may provoke aseptic inflammation while degrading
in vivo
hindering, resulting
in a complete regeneration, facilitating the formation of fibrotic tissue and inhibiting the
development of COL1, the main component of tendon ECM [
122
–
124
]. It is hence thought
to overcome the undesirable effect of released acidic by-product, during the degradation
process of the scaffolds, to modify scaffold chemical properties by incorporating basic
molecules able to neutralize acidic compounds, reduce inflammation, and enhance tissue
regeneration [
98
] (Table 1). For example, Shen et al., fabricated shell-core structured fibers of
CTS/PLGA with acid-neutralizing capability by coating the core surface of PLGA aligned
fiber with a layer of the alkaline CTS using the coaxial electrospinning [
98
]. Indeed, even if
PLGA represents an FDA-approved biomaterial and provides sufficient control of degrada-
tion [
125
,
126
], by this study, the pH decrease resulted from an 8-week PLGA degradation
period that was impeded by the CTS layer maintaining the degradation medium pH at 6.
Moreover,
in vivo
experiments conducted subcutaneously on mice for two and four weeks,
showed a significant decrease in inflammatory cell recruitment and in the formation of
FBGCs within the CTS/PLGA fibers. Therefore, this work reported the acid-neutralizing
role of the chitosan-coating layer on lightening the inflammatory responses due to the
PLGA by-products’ acidic degradation [
98
]. Jiang et al., conducted a study to understand
which of these factors, namely the type of degradation by-products, material debris, and
the acidic environment, are responsible for the subsequent immune reactions. The results
showed that water-insoluble PLLA debris as well as water soluble PLLA/Polyethylene
glycol (PEG) (1:1) debris are responsible for the regulation of the phagocyte activation
and the subsequent tissue responses, as measured by reactive oxygen species production
and inflammatory cell recruitment in both
in vitro
and
in vivo
models [
127
]. The findings
that activated macrophages can phagocytose PLLA/PLGA particles [
128
] and that particle
phagocytosis can induce the macrophages activation [129] backed up these observations.
4.1.3. Effect of Environment Scaffold Subjection on Immune Cells
Mechanical and Electromagnetic Stimuli Influence on Immune Cells
Depending on the implantation site (i.e., presence of blood vessel, active joint loading
or muscles) and tissue target of the regeneration, the scaffold may be subjected to dynamic
or cyclical loading, resulting in cell dynamic or cyclical strain [
130
]. Mevoy et al. measured

Cells 2022,11, 266 15 of 40
the impact of cyclical pressure on cytokine/chemokine production in cultured human
macrophages, finding that all levels of cyclical pressure tested (17–138 kPa) resulted in an
increase in pro-inflammatory cytokines (TNF-a, IL-6, IL-1
β
) compared to controls [
131
].
Researchers routinely expose electrospun scaffolds to mechanical stimuli in order to study
the immunomodulatory consequence on the seeded cells (Table 1). Schoenenberger et al.
studied the response of macrophages seeded on PCL nanofibers with aligned or random
orientation, under conditions of mechanical load and unload (static). Macrophages were
exposed to static (1% constant strain) or dynamic loading (7% cyclic strain at 1 Hz) for 8 h,
followed by a 16 h quiescent period. Rather than the fibers’ alignment modulatory effect
on macrophages, dynamic loading led to an upregulation of CCR7 (M1 marker), meaning
the macrophage polarization through the pro-inflammatory phenotype [
93
]. Similarly,
Bonito et al. assessed the effect of cyclic strains (0%, 8% and 14% strain at 0.8 Hz) on human
peripheral blood mononuclear cells (hPBMCs) seeded on electrospun chain-extended
(CE) ureido-pyrimidinone (UPy) modified PCL (CE-UPy-PCL) sheets with two different
fiber diameters, 4
µ
m or 13
µ
m. They underlined a strain-dictated modulation of the
inflammatory response (high strains addressed a pro-inflammatory condition) minimally
affected by the different fiber diameters of the electrospun scaffolds [
96
].
In vitro
and in turn
in vivo
experiments revealed that macrophages are mechanically responsive and change
their lineages in response to mechanical stimuli. Mechanical stimuli in the form of tensile
micromechanical strains, hydrostatic cyclic pressure, and compressive strains around the
implant site further accelerate macrophage inflammatory cytokine synthesis [
130
], therefore
the investigation of the macrophages’ response to those mechanical stimuli is fundamental
for the design of an ideal scaffold [130].
Uniaxial and biaxial loading are the most common stimuli for tissues of the mus-
culoskeletal apparatus (namely muscle, ligaments, and tendons). In detail, tendons and
ligaments are subjected to uniaxial strain in the 5–16% range [
130
]. In this field, Ballotta et al.
applied 7 and 12% cyclic uniaxial strains on hPBMCs seeded on a 25 mm by 5 mm 2D PCL-
based tissue scaffold strip. Flexcell™ was used to apply mechanical strains at a frequency
of 0.8 Hz for 7 days. They performed gene expression and immunohistochemistry analysis
to study the expression of immune response markers in response to mechanical strain.
Their findings indicated that a moderate amplitude of 7% mechanical strain facilitated
hPBMCs’ polarization through the anti-inflammatory M2 phenotype and enhanced the
expression of MCP-1, IL-6, IL-10, and MMP-9. In contrast, a 12% strain induced hPBMCs to
acquire the M1 pro-inflammatory phenotype [95].
In a different way, another external trigger of the immune response has been found in
the use of pulsed electromagnetic field (PEMF). These magnetic impulses can modulate
inflammatory response in macrophages at low frequency. Vinhas et al. explored the
potential modulatory effect of this technology in macrophage–human tendon cells (hTDCs)
communication. They applied a magnetic stimulation regimen at 5 Hz, 4 mT, and 50% duty
cycle for 1 h, using a magnetotherapy device. The PEMF resulted to influence a macrophage
pro-regenerative phenotype (increased Arg-1, MRC-1, and Singlec-1 M2-like markers) and
favors the synthesis of anti-inflammatory mediators (IL-6 and TNF
α
downregulation) [
132
].
How Oxygen Concentration Influences Immune Cells
A functionalized scaffold can provide in loco also changes in the local oxygen concen-
tration. In detail, hypoxia can be used in cooperation with other factors to address and boost
the tendon healing since tendons and ligaments are poorly vascularized tissues [
133
], with
an oxygen consumption 7.5 times lower than skeletal muscles [
134
]. Assuming that skeletal
muscle oxygenation is about 2–5% O
2
(7.5 to 31 mmHg) [
135
,
136
], lower oxygen concentra-
tion appears to be crucial for a successful tendon recovery [
34
]. In addition, macrophages,
in hypoxic conditions, accumulate and release pro-healing and pro-angiogenic factors such
as VEGF and platelet-derived growth factor (PDGF), as well as enzymes such as cyclooxyge-
nase 2 (COX-2). Hypoxia inducible factor (HIFs) 1 and 2, transcriptional factors, are thought
to mediate macrophage responses to hypoxic environments. Although macrophages play a

Cells 2022,11, 266 16 of 40
beneficial role in promoting vascularization of embedded scaffolds in a hypoxic wound
setting, it should be noted that the hypoxic environment found in cancerous tissue actively
recruits tumor-associated macrophages (TAMs) [26].
4.2. Immuno-Induction of Scaffold on Stem Cells
As described previously, stem cells, including MSCs [
52
,
61
,
137
–
139
] and amniotic-
derived stem cells [
17
–
19
,
140
–
143
], have been widely used in tendon TE where they
have shown teno-differentiative abilities allowing them to be a promising key element
to be used in tendon regeneration [
34
]. Although they have the ability to differentiate
towards the tenogenic lineage
in vitro
and
in vivo
, MSCs and amniotic-derived stem
cells exhibited high immunomodulatory properties which in turn could enhance ten-
don
regeneration [34,82,87,143–146]
. Given the highly dynamic property of innate immune
response and the immunomodulatory properties of stem cells, the combination of these
last as well as their paracrine factors with tendon-like scaffold could represent an ideal
microenvironment to module immune cells and the regeneration process within the tendon
injury site. For this reason, new strategies must be developed by providing a suitable
microenvironment for the tissue in regeneration, allowing an improved stem cell recruit-
ment and their differentiation together with angiogenic reactions, avoiding any adverse
inflammatory response [24].
The electrospun scaffolds can be designed not only to direct immune cell behavior
and avoid scaffold rejection but can be also fabricated to tune and boost the immunomod-
ulatory response of stem cells, which can in turn activate a new signaling cascade lead-
ing to the resolution of the damaged tendon tissue (Figure 5). Although many efforts
have been made to assess the effect of physical and chemical properties of electrospun
scaffolds on stem cell proliferation, migration and differentiation towards the tenogenic
lineage [17–19,29,33,34,40,147]
and their effects on regulating the immunomodulatory prop-
erties of stem cells must still be studied in depth. While researchers have focused their
attention on the effect of intrinsic and extrinsic properties of electrospun scaffolds on
affecting the cellular behavior of immune cells, few studies have evaluated the immuno-
induction potential of this type of scaffold on stem cells by assessing mainly fiber alignment
and diameter size as well as their exposition to mechanical stimuli.
4.2.1. Intrinsic Properties of the Scaffold on Stem Cells
Topography Effect on Stem Cells: Fiber Alignment and Diameter Size
Stem cells belonging to bone marrow (BM) and adipose-derived (AD) MSCs together
with AECs have been used to assess the effect of scaffold fiber alignment and diameter
size on tuning their innate immunomodulatory properties [
16
,
17
,
32
,
84
,
85
]. The conducted
studies revealed aligned topography rather than oriented fibers together with small fiber
diameter size in the micrometer range of the electrospun scaffolds influenced the im-
munomodulatory properties of stem cells by regulating the gene and protein expression of
anti-inflammatory cytokines compared to the pro-inflammatory ones, and by activating the
mechanotransduction pathways during tendon regeneration (Table 2) [18,33,83–85].
Su et al., fabricated three electrospun PLC scaffolds with randomly oriented (R),
aligned (A) and mesh-like (M) electrospun fibers (EFs) and they assessed their effect
on the paracrine function of ADMSCs [
83
]. The obtained results showed that oriented
AEFs and MEFs enhanced the expression of PGE
2
, iNOS, and VEGF within ADMSCs
compared to those engineered within the REFs [
83
]. Moreover, the conditioned media
(CM) obtained from the culture of ADMSC-MEFs and ADMSC-AEFs exhibited potent anti-
inflammatory responses, characterized by elevated expression of IL-10 when cultivated with
macrophages cells compared to REFs [
83
]. Similarly, in a study conducted by Wan et al., they
demonstrated that ADMSCs engineered within PLLA electrospun scaffold with aligned
fibers exhibited higher expression of immunomodulatory factors including PGE
2
, COX-2,
TGF-
β
, macrophage colony stimulating factor (M-CSF) and TSG-6 [
84
]. TSG-6 acts as an
inhibitor of the TLR 2-nuclear factor-B (NF-B) signaling in resident macrophages, reducing

Cells 2022,11, 266 17 of 40
the typical activation of these cells into the phenotype M1 [
148
]. It also prevents neutrophil
infiltration by interfering with the interaction of cell-surface glycosaminoglycans with a
variety of chemokines from the CC and CXC subfamilies [
149
]. Regarding COX-2, it is a
necessary enzyme to produce PGE
2
, a potent immunosuppressive mediator, which can
modulate the phenotype of macrophages from M1 to M2 [150].
Table 2.
Overview of the influence of different electrospun scaffolds properties on the immunomodu-
latory properties of stem cells.
Material Stem Cell Type Propriety Outcomes Reference
PCL Rat ADMSCs
Randomly oriented,
aligned and mesh-like
electrospun fibers
↑gene expression of PGE2, iNOS, and
VEGF within ADMSCs engineered within
aligned and mesh-like fibers
[83]
PLLA Human ADMSCs
Randomly oriented and
highly aligned
electrospun fibers
↑gene expression of COX-2, TGF-β,
TSG-6, and M-CSF in ADMSCs cultured
within aligned fibers
↑protein expression of COX-2 and TSG-6
and
↑
secreted levels of PGE
2
in ADMSCs
on aligned fibers
[84]
PLGA Ovine AECs
Electrospun PLGA
scaffolds with two
different diameter size
(1.27 and 2.50 µm)
↑gene expression of IL-4 and IL-10 and ↓
gene expression of IL-12 and IL-6 within
small fiber diameter size (1.27 µm)
[18]
PCL Human ADMSCs
Electrospun
PCL-DT-NPs yarns
cultivated under static
and magnetic
stimulation conditions
↑gene expression of MMP-1, MMP-2,
MMP-3, TIMPs, IL-10, and IL-4 with ↓
gene expression of IL-6 and COX-2 under
magnetic stimulation condition
[33]
PCL: polycaprolactone; PLLA: poly(L-lactide); PLGA: poly(lactic-co-glycolic acid); ADMSC: adipose-derived
mesenchymal stem cells; AECs: amniotic epithelial stem cells; ↑: increase; ↓: decrease.
Recently, it has been demonstrated that also fiber size of electrospun scaffolds with
highly aligned fibers affect the immunomodulatory profile of stem cells. For instance,
El Khatib et al. demonstrated that changing diameter size of aligned electrospun PLGA
scaffolds alters the interleukin profile of AECs [
18
]. The obtained results revealed that AECs
seeded on electrospun PLGA scaffolds with small fiber diameter size (1.27 µm) expressed
high levels of pro-regenerative, anti-inflammatory cytokines (IL-10) with favorable IL-12/IL-
10 ratio compared to larger fiber size (2.50
µ
m), which induced on AECs’ high expression of
pro-inflammatory cytokines (IL-12 and IL-6) [
18
]. The importance of lowering IL-12/IL-10
ratio appears to influence positively the inflammatory response of AECs by supporting
their transition from an inflammatory to reparative phase, demonstrating a macrophage
skewing towards the M2 pro-regenerative phenotype, as previously demonstrated by
Manuelpillai et al. [
151
] in hAECs xeno-transplanted liver, and Mauro et al. [
82
] and
Barboni et al. [
140
] observations in oAECs allo-transplanted and hAECs xeno-transplanted
tendons, respectively.
4.2.2. Effect of Environment Scaffold Subjection on Stem Cells
Mechanical Stimuli Influence on the Immunomodulation of Stem Cells
The remodeling of tendon ECM by the activity of MMP and TIMPs can be improved
by applying mechanical stimuli. MMP and TIMPs are key elements for tendon tissue
repair and maintaining their activities in balance is indispensable for healthy tendon
homeostasis [
152
]. Tomás et al., fabricated electrospun PCL yarns incorporated with DT-
nanoparticles (DT-NPs), engineered with human ADMSCs (hADMSCs) and cultivated
under static and magnetic stimulation culture (Table 2) [
33
]. The obtained results showed
that engineered hADMSCs-PCL-DT-NPs yarns, cultivated under magneto-mechanical

Cells 2022,11, 266 18 of 40
stimulation, exhibited high expression levels of MMPs including MMP-1, MMP-2, and
MMP-3 together with TIMPs when compared to engineered cells cultivated under static
condition. Moreover, the analysis of pro- (IL-6 and COX-2) and anti- (IL-4 and IL-10)
inflammatory cytokines showed that hADMSCs within PCL-DT-NPs yarns upregulated
the anti-inflammatory cytokines’ gene expressions while downregulating those of pro-
inflammatory ones, with a notable boosted expression of IL-4 and IL-10 under magneto-
mechanical stimuli [33].
4.3. Biological Strategies to Enhance the Immunoregenerative Potential of the Scaffolds
Synthetic and natural biomolecules could represent an advanced strategy to modulate
the host response of the damaged tissue due to the ability to tune their delivery timing at
specific phases of inflammation and repair
in vivo
(Table 3). For instance, local releases
may be used to create a gradient of a specific compound necessary to recruit monocytes and
macrophages at a specific time within the injury site [
30
]. To this end, biological strategies
have been developed with the aim to directly deliver bioactive molecules, chemokines, and
nanoparticles, or incorporate them within the electrospun scaffolds providing a feasible
microenvironment able to boost the immunomodulatory properties of transplanted or
recruited cells within the injury or implantation site. The functionalization of the scaf-
folds with bioactive molecules can be considered as an intrinsic feature since it might be
performed during or immediately after scaffold preparation.
Table 3.
Overview of the influence of bioactive molecules on the immunomodulatory properties of
tenocytes, stem and immune cells.
Bioactive Molecule Scaffold Material Cell Type Outcomes in the Studied Cell Type Reference
NSAIDs PELA Macrophages ↓inflammatory response and ↓TA [85]
IL-4 CE-UPy-PCL Macrophages ↑IL-10, TGF-β1 and MMP-9
↓IL-6 [2]
IL-4 PCL Macrophages ↑M2 macrophage markers (Arginase I, CD206 . .. ) [153]
25-hydroxyvitamin D3PCL Macrophages ↓TNF-a, IL-6 and ↑IL-4, IL-10 [154]
Ibuprofen PLA Macrophages ↓TNF-αexpression and collagen III deposition [155]
Mesenchymal stromal
exosomes PEF BM Macrophages
↑CD206+ M2 macrophages and the concentration of
IL-4, IL-10 and IL13
↓concentration of TNF-αand IFN-γ
[156]
IL-4 plasmid-loaded
liposomes (aL/p) MSaP BM Macrophages ↑levels of IL-10 and TGF-β
↓levels of IL-1 and TNF-α[157]
Melatonin PCL Human BMSCs Inhibition of macrophage (CD68-positive cell)
accumulation at the tendon-to-bone interface. [158]
MSCs-derived ECM PCL/SF Human BMSCs
In vitro: ↑M2 macrophage polarization and ↓IL -1β,
IL-6, CXCL11, IL-10, IL-1R2 and TGF-β1
In vivo: ↓FBR, thinner fibrotic capsule formation and
↑M2 macrophage polarization
[159]
bFGF PLLA Human vaginal fibroblasts ↑concentration of TGF-β1 and ↓concentration of
TNF-α[160]
IFN-γSF/PLGA Human BMSCs ↑transcription levels of COX-2 and IDO
↓transcription levels of TNF-α[161]
OLE PHA Human HaCaTs ↓concentration of IL-1, IL-6, IL-8 and TNF-α[162]
IFN-γand TNF-αNo scaffold Human BMSCs ↑gene expression of IDO, iNOS, IL-6, COX-2 and
VCAM-1 [163]
PRP No scaffold Human tenocytes ↑concentration of VEGF, RANTES and HGF
↓gene expression of IL-6, IL-6R, and IL-8 [164]
HGF No scaffold Tendon fibroblasts
↑concentration of MMP-2 and MMP-9, α-SMA,
TIMP-1, VEGF and IL-10
↓gene expression of IL-6 [165]
BMP-12 No scaffold Human ASCs ↑concentration of VEGF, MMP1, MMP8 and IL6 [166]
PCL: polycaprolactone; CE-UPy-PCL: ureido-pyrimidinone (UPy)-modified chain extended polycaprolactone;
PLLA: poly(L-lactide); PLA: polylactide; PLGA: poly(lactic-co-glycolic acid); PELA: poly(lactic acid-co-Ethylene
glycol-co-Lactic Acid); PEF: fibrous polyester; MsaP: microsol electrospun fiber scaffold; BMM
Φ
s: bone marrow
macrophages; BMSCs: bone marrow mesenchymal stem cells; HaCaT: human dermal keratinocytes; bFGF: basic
fibroblast growth factor; OLE: olive leaf extract; PRP: platelet-rich plasma; HGF: hepatocyte growth factor;
↑
:
increase; ↓: decrease.

Cells 2022,11, 266 19 of 40
Significant efforts have been also made to modify the synthetic biomaterial scaffolds
with biological properties such as cytokines or anti-inflammatory drugs for delivering at
the injury site or in a surface coated form [100].
Although these combinational strategies have been widely used for different TE appli-
cations, only a few studies have addressed the use of scaffolds with aligned fibers which can
simulate the tendon-like structure of the ECM for immunoengineering [
154
,
158
]. One of these
investigations functionalized the electrospun aligned fibers’ scaffold with 25-hydroxyvitamin
D3 (25(OH)D
3
) [
154
]. This molecule has aroused great interest in the design of medical scaf-
folds that could modulate the immune response [
167
,
168
].
In vitro
studies highlighted the
role of vitamin D3 in the reduction of pro-inflammatory cytokines’ expression together with
the increased production of anti-inflammatory ones [
167
–
169
]. Chen et al., evaluated the host
responses using 25(OH)D
3
eluting radially aligned electrospun PCL nanofiber scaffolds subcu-
taneously implanted in humanized mice [
154
]. The results showed that rather than significantly
reducing the production of pro-inflammatory cytokines (TNF-a, IL-6), the production of anti-
inflammatory cytokines (IL-4, IL-10) was boosted [
154
]. Indeed,
in vitro
and
in vivo
studies
suggest that 25(OH)D
3
exhibited a suppressive role on the expression of pro-inflammatory
cytokines while raising that of anti-inflammatory cytokines [
167
–
169
]. For instance, Zhang et al.
showed how 25(OH)D
3
inhibited the production of the pro-inflammatory cytokines IL-6 and
TNF-
α
from monocytes/macrophages, by upregulating mitogen-activated protein kinases
(MAPKs) phosphatase-1 expression and suppressing p38 activation [
169
]. Another interesting
molecule, able to be part of the immune response modulation, is melatonin. This hormone is
largely secreted by the pineal gland, and has numerous biofunctions such as anti-inflammatory,
antioxidation and immunomodulation properties [
170
]. Recently, melatonin has shown to
inhibit the expression of pro-inflammatory markers in macrophages [
171
]. In order to obtain
more information about this type of modulation, Song et al. cultured human bone marrow
mesenchymal stem cells (hBMSCs) with melatonin-loaded aligned PCL electrospun fibrous
scaffolds and tested them
in vivo
in a rat acute rotator cuff tear model. During the early
healing phase, melatonin-PCL electrospun scaffolds could significantly inhibit M1 macrophage
(CD68-positive cell) accumulation at the tendon-to-bone interface and improved the collagen
fiber organization of the ECM [158].
Although most of the studies describing the incorporation of bioactive molecules were
performed on randomly oriented fibers [
85
,
153
,
155
,
157
,
161
,
165
,
171
,
172
], which do not mimic
tendon structure, their description can be of great interest since they could represent novel
immunoinformed strategies to develop functionalized tendon biomimetic scaffolds aimed
at boosting scaffold’s efficacy and its regenerative potential [
85
,
153
,
155
,
157
,
161
,
165
,
171
,
172
].
Within these investigations, a wide range of biomolecules used can be found. The release of
factors (e.g., IL-4, IL-10, steroids) overwhelming the native signaling and directing macrophage
polarization is a well-known and targeted methodology to control the immune response in
an immunomodulated regenerative biomaterial [
173
,
174
]. In particular, the encapsulation of
growth factors, gene delivery vectors, or small molecule drugs (e.g., steroids) in triggered release
platform is the most used technique to achieve the above mentioned release of factors [175].
McWhorter et al. found that the morphological elongation of macrophages combined
with M2 inducing cytokines (IL-4, IL-13) increased M2 polarization, meaning that bio-
physical cues directly presented by biomaterials may be used to complement the effects
of factors already present in the native environment, in addition to directing polariza-
tion [
176
]. The biological response can be modulated to optimize repair by reducing the
inflammatory response engineering material properties and biomolecule delivery [
30
]. To
condition local macrophages to a specific phenotype, polarizing cytokines can also be
released. For example, Qian et al., incorporated the anti-inflammatory cytokine IL-4 within
silk fibroin-functionalized electrospun PCL with randomly oriented nanofibers using the
layer-by-layer assembly technique that led
in vitro
to M2 macrophage polarization char-
acterized by the detection of CD206 and Arginase I markers in a murine subcutaneous
model [
153
]. Moreover, Bonito et al., developed an electrospun CE-UPy-PCL scaffold
with randomly oriented fibers, functionalized with a UPy-modified heparin binding pep-

Cells 2022,11, 266 20 of 40
tide (UPy-HBP) to immobilize IL-4 through the heparin binding domain. Therefore, after
cultivating human PBMCs on the IL-4 functionalized scaffolds, the macrophages demon-
strated to support an anti-inflammatory environment characterized by IL-10 upregulation
at day 3, IL-6 downregulation, and TGF-
β
1 and MMP-9 overexpression at both day 3
and 7 after
in vitro
culture [
172
]. A further factor that overwhelms native signaling is the
pro-inflammatory cytokine IFN-
γ
, which has been shown to induce the MSCs to secrete
factors such as COX-2, PGE
2
, and indoleamine 2,3-dioxygenease (IDO) [
177
]. In the study
carried out by Kim et al., hBMSCs were cultured on electrospun silk fibroin scaffolds (SFN)
or PLGA nanofiber scaffolds, both of them randomly oriented, and treated with human
IFN-
γ
[
161
]. In both situations, IFN-
γ
significantly increased the transcription levels of the
immunomodulatory cytokines COX-2 and IDO, as well as the secretion of IL-10 compared
with the control without scaffold. Instead, on the opposite side, it suppressed the secretion
of TNF-αby the splenocytes [161].
Another study was carried out
in vitro
by Wang et al., in which they grafted basic
fibroblast growth factor (bFGF) on the surface of electrospun PLLA scaffold with randomly
oriented fibers to improve its hydrophilicity and assess its immunomodulatory poten-
tial [
160
]. A hydrophilic polypeptide, bFGF can stimulate angiogenesis, speed wound
healing and tissue repair, enhance tissue regeneration and boost collagen production [
178
].
The creation of a constant bFGF release from the fabricated scaffold increased the anti-
inflammatory-related cytokine TGF-
β
1 within human vaginal fibroblasts while decreased
the concentration of TNF-α[160].
Traditional medicine has used olive tree products as botanical medications and food
additives [
179
]. Olive leaf extract (OLE) has been studied for a variety of uses, including
an anti-inflammatory drug, and it is well-known for being a good source of antioxidants,
bioactive chemicals, and even polyphenols [
179
]. De La Ossa et al., investigated with
randomly oriented electrospun polyhydroxyalkanoate (PHA) scaffolds functionalized
with OLE, taking into consideration its polyphenols, more concretely oleuropein, as well
as luteolin-7-O-glucoside and aspigenin-7-O-glucoside in lower concentrations. These
scaffolds were cultured
in vitro
with human dermal keratinocytes (HaCaT cells) for a
period of 24 h. When released by PHA fiber meshes, OLE has been shown to have diverse
immunomodulatory effects
in vitro
, switching from pro-inflammatory to anti-inflammatory
environments by downregulating IL-1, IL-6, IL-8 and TNF-
α
, and even stimulating defensin
in the case of polyhydroxybutyrate (PHB)/poly(hydroxyoctanoate-co-hydroxydecanoate)
(PHB/PHOHD) scaffolds [162].
A different approach for a scaffold functionalization was performed by Xi et al., where
randomly oriented microsol electrospun fiber scaffolds (MSaP) were engrafted with IL-4
plasmid-loaded liposomes (aL/p) in order to assess
in vitro
their immuno-inductive proper-
ties. The functionalized scaffolds were cultured through a trans-well system with BMM
Φ
s
for a period of 7 days. The cells cultured on MSaP-aL/p groups exhibited a progres-
sively decreased expression of pro-inflammatory genes IL-1 and TNF-
α
over time. Instead,
the gene expression of anti-inflammatory gene IL-10 and the transforming growth factor
(TGF)-βincreased within cells cultured on MSaP-aL/p compared with the control [157].
Nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen and celecoxib,
can reduce TA formation by reducing inflammation and in turn by regulating the heal-
ing microenvironment, as showed by Zhang et al. who reported the loading of NSAIDs
into poly(lactic acid-co-ethylene glycol-co-lactic acid) (PELA) EFMs [
85
]. Furthermore,
ibuprofen-loaded electrospun PLA EFMs limited
in vivo
macrophage adhesion, prolifer-
ation, and infiltration by inhibiting TNF-
α
expression and collagen type III deposition,
and reduced the inflammation and granuloma formation in the area surrounding the
tendon [155].
Exosomes, known as membrane-enclosed extracellular vesicles (EVs) that carry pro-
teins and nucleic acids, might represent another type of delivery system of bioactive
molecules [
180
]. Su et al. developed biofunctional scaffolds by fixing mesenchymal
stromal exosomes (Exo) to electrospun randomly oriented fibrous polyester materials

Cells 2022,11, 266 21 of 40
(PEF) [
156
]. The fabricated scaffolds were examined in skin injury models of sham mice
to understand the immunomodulatory effects of mesenchymal stromal exosomes (Exo)
in vivo
[
156
]. MSC-associated immuno-moderation activity supports the M2-like pheno-
type of macrophages, population of T
reg
and immunological Th2 responses [
181
]. In fact,
Su et al., demonstrated that Exo-PEF increased the number of immunomodulatory CD206+
M2 macrophages by a factor of one. On day 7, Exo-PEF also increased the number of T
reg
cells by more than 3-fold compared to untreated wounds, as well as the proportion of
T
reg
cells secreting the anti-inflammatory cytokine IL-10. Furthermore, compared with
untreated mice, Exo-PEF promoted the secretion of IL-4, IL-10, and IL-13 and decreased
the pro-inflammatory chemokines TNF-
α
and IFN-
γ
[
156
]. Another example of exosome
involvement was shown by Chamberlain et al., which generated M2-like macrophages,
avoiding the usage of MSCs directly, by using exosomes isolated from MSCs and creating
exosome-educated macrophages (EEMs) [
182
,
183
]. For example, the researchers next com-
pared the effects of EEMs on mouse Achilles’ tendon rupture to normal tendon healing,
MSCs, and EVs. Exogenous delivery of EEMs directly into the wound enhanced tendon
mechanical characteristics, reduced inflammation, and accelerated angiogenesis, according
to Chamberlain et al., whereas treatment with MSC-derived EVs alone was less beneficial
while reducing the M1/M2 ratio [
182
]. Moreover, Shen et al. assessed the effect of adipose
stem cells’ (ASCs) EVs on early tendon healing using a mouse Achilles tendon injury and
repair model [
184
]. Tendon healing was evaluated in nuclear factor-kB (NF-kB) luciferase
reporter mice, up to 7 days after surgery. Following tendon repair, NF-kB activity increased
by more than threefold [
184
–
186
], the reaction was effectively reduced by using EVs from
primed but not naïve ASCs. Furthermore, in repaired tendons, the pro-inflammatory genes
Il-1b and IFNG were both markedly elevated, whereas primed, but not naive ASC EVs
reduced the response [
184
]. Several studies have demonstrated the effect of immunomodu-
latory properties of MSCs on reducing the inflammasome in macrophages by modulating
macrophage polarization [
159
,
187
–
191
]. However, only the study conducted by Dong
et al. implied the functionalization of randomly oriented electrospun PCL/silk fibroin
scaffolds with decellularized ECM derived from hBMSCs (PCL/SF-ECM) (Table 3) [
159
].
In this study, the role of bioactive molecules contained within the PCL/SF-ECM scaffold
was assessed on modulating macrophages’ polarization for tendon applications. The fab-
ricated scaffold enhanced M2 macrophage polarization and reduced the expression of
multiple cytokines (IL -1
β
, IL-6, CXCL11, IL-10, IL-1R2 and TGF-
β
1)
in vitro
[
159
]. TGF-
β
1
is a profibrotic factor that stimulates fibroblast recruitment and in turn collagen secretion
through the Smad3 pathway [
192
,
193
]. Indeed, probably because of scaffold inhibitory
effect on TGF-
β
1 expression, the results of rat subcutaneous implantation showed a lower
FBR, a thinner fibrotic capsule formation, and a higher M2 macrophage phenotype po-
larization [
159
]. Furthermore, Aktas et al., used a rat Achilles segmental defect model
to assess the
in vivo
healing benefits of TNF-
α
-primed MSCs [
194
]. Rat Achilles tendons
were damaged and then repaired using a PLGA scaffold alone, an MSC-seeded PLGA
scaffold, or a TNF-
α
-primed MSC-seeded PLGA scaffold. Samples were analyzed two and
four weeks after the injury. MSCs boosted the production of IL-10 while decreasing the
inflammatory factor IL-1a while primed MSCs decreased the production of IL-12 and the
number of M1 macrophages simultaneously increasing the percentage of M2 macrophages
and IL-4 synthesis. When TNF-
α
-primed MSCs were delivered via 3D PLGA scaffold,
macrophage polarization and cytokine production were regulated, enhancing the more
regenerative MSC-induced healing response [194].
Apart from the studies regarding the effects of the bioactive molecules on improving
the teno-inductive potential of electrospun scaffolds, other investigations concerning the
use of different kinds of biocompounds to improve the modulatory effects of the immune
system in tenocytes and stem cells were performed [
163
–
166
]. As previously mentioned,
some pro-inflammatory molecules such as IFN-
γ
induce the secretion of immunomodu-
latory proteins, such as COX-2, PGE
2
or IDO [
177
] in the MSCs. An experiment in which
BM-MSCs exposed to IFN-
γ
and TNF-
α
was performed. At low concentrations of these

Cells 2022,11, 266 22 of 40
cytokines, there was an upregulation of the immunomodulatory genes IDO, iNOS, IL-
6, COX-2 and VCAM-1. There was also an increase in the expression of MHC-II and
CD40 [
163
]. Another molecule with promising therapeutic uses is platelet-rich plasma
(PRP), which is renowned for influencing the early healing response by secreting a variety
of signaling cytokines that regulate inflammation and angiogenesis at the same time, as
well as cell migration and proliferation [
195
]. In a research study conducted by Andia et al.,
the immunomodulatory effects of PRP were assessed
in vitro
on tenocytes exposed with or
without IL-1
β
to induce an inflamed phenotype [
164
]. The treatment with PRP reduced the
expression of pro-inflammatory interleukins including IL-6, IL-6R, and IL-8 in the IL-1
β
exposed cells. The secretion of IL-6, IL-8, and monocyte chemoattractant protein-1 was
also reduced after PRP treatment, whereas VEGF increased 2-fold. In tendinopathic cells,
regulated upon activation, normal T cell expressed and secreted (RANTES), representative
of C–C chemokines, grew 10-fold and hepatocyte growth factor (HGF) increased 21-fold,
while in normal cells increased 2.3-fold [
164
]. Another bioactive molecule was studied by
Zarychta-Wi´sniewska et al., which treated human ASCs (hASCs) with Bone morphogenic
protein 12 (BMP-12) in order to determine
in vitro
its tenogenic modulation, as well as its
induced immunomodulatory on hACSs [
166
]. The BMP-12 belongs to a protein group that
can induce ectopic formation of tendon-like structures and improve healing parameters in
injured tendons [
196
]. The results showed that BMP-12 induced
in vitro
tenogenic differen-
tiation of hASCs, and stimulated VEGF, MMP1, MMP8 and IL6 secretion within the cells
substantially. Instead, no effect was detected in the transcription levels of EGF, IL-10, TGF-
β
and MMP-13 [166]. In a different scenery, Zhang et al. performed an experiment in which
tendon fibroblasts were cultured in a conditioned medium from tendon stem cells enriched
with a gradually increased concentration of HGF [
165
]. In these circumstances, there was
an upregulation of MMP-2 and MMP-9,
α
-SMA, TIMP-1 and VEGF in the ECM, as well as
the anti-inflammatory cytokines such as IL-10. On the other hand, a downregulation of the
pro-inflammatory cytokines such as IL-6 was observed. These results were verified both
in vitro
and
in vivo
, with a more notable effect with the higher concentrations of HGF [
165
].
Another method to functionalize scaffolds with bioactive molecules is implicated by
their encapsulation or loading NPs to control their delivery and release to modulate the
inflammatory response that might occur at the injury site [
197
]. NPs have been widely used
in different applications including the production of sensors, materials construction, as well
as in the biomedical field for different applications such as drugs delivery, vaccine adjuvants
or catalysts to boost chemical reactions [
198
]. The use of NPs in regenerative medicine has
been increasing over the years, thanks to the improvements of the synthesis techniques,
as well as the new FDA approved materials used [
199
]. In some cases, NPs themselves
can exhibit immunomodulatory potential. A study concerning this topic in a tendon-
like environment was performed by Vinhas et al., where they developed magnetically
assisted cell sheets (magCSs) by using hTDCs with magnetic NPs (MNPs). MagCSs were
in vitro
subjected to IL-1
β
to induce an inflammatory-like environment and then were
exposed to a PEMF. The results showed that under these conditions, the levels of pro-
inflammatory factors IL-8, IL-1
β
, TNF-
α
, and IL-6 decreased, whereas the transcription
levels of anti-inflammatory factors IL-10 and IL-4 increased. Thus, PEMF appeared to
successfully restore anti-inflammatory factor levels in inflammatory conditions, considered
crucial for the tendon healing process [
200
]. A similar study conducted by the same
research group developed magnetic membranes of a polymeric blend of PCL and starch
(S) with iron oxide nanoparticles (magSPCL), which were cultured
in vitro
with hTDCs
and exposed to PEMF [
201
]. The levels of the pro-inflammatory molecules TNF-
α
, IL-6,
IL-8 and COX-2 decreased, while the expression of anti-inflammatory cytokines (IL-4 and
IL-10) increased. The results indicate that magSPCL exposed to PEMF can successfully
suppress the expression of NF-
κ
B, a transcription factor that regulates and coordinates
the expression of various pro-inflammatory genes and mediators, including cytokines,
chemokines, adhesion molecules, immunoreceptors and growth factors [
202
]. Indeed,
these conditions were used to assess the effect of magnetic membranes on macrophages.

Cells 2022,11, 266 23 of 40
These last showed a more elongated shape, associated with a pro-healing phenotype, when
cultured under the above-described conditions. Moreover, macrophage behavior was
confirmed by the increased expression of the surface markers CD16
+
, CD169
+
and CD206
+
,
indicating their polarization towards the M2 phenotype [201].
In addition to the immunomodulatory effects of nanoparticles under specific condi-
tions, other studies have focused on their role in the delivery control of bioactive molecules
and chemical compounds. In a study conducted by Kang et al., porous PLGA microspheres
(PMSs) were synthetized with the immobilization of heparin-dopamine (Hep-DOPA),
and platelet-derived growth factor (PDGF) (PDGF/Hep-PMSs) to
in vitro
examine the
inflammatory responses on LPS-stimulated rabbit tenocytes [
203
]. The results showed
a high degree of immunomodulation characterized by suppressing the mRNA levels of
six pro-inflammatory cytokines MMP-3, MMP-13, COX-2, IL-6, TNF-
α
and A Disintegrin
and Metalloproteinase with ThromboSpondin motif (ADAMTS-5), while increasing the
mRNA levels of anti-inflammatory cytokines IL-4, IL-10, and IL-13 [
203
]. A comparable
study concerning the same type of NPs was performed by Jeong et al., where they de-
veloped simvastatin-loaded porous PLGA microspheres (SIM/PMSs) on (LPS)-treated
tenocytes [
204
]. Simvastatin belongs to the drug class of statins (used to lower blood
cholesterol), and exhibits anti-inflammatory properties accompanied with side effects when
administrated at high doses [
205
]. The research was conducted to assess (LPS)-treated
tenocytes’ immune response at different concentrations of SIM/PMSs and showed that the
outcome of the microspheres had a dose-dependent decrease in the mRNA levels of MMP-3,
COX-2, IL-6, and TNF-
α
, with stronger effect at 5 nM compared to 1 nM. Additionally, in
an
in vivo
model of RC tendinopathy there was a slight increase in the mRNA levels of the
anti-inflammatory cytokines IL-4, IL-10, and IL-13 [
204
]. In a further study using the PLGA
material, Jong Choi et al. fabricated lactoferrin-immobilized, heparin-anchored, PLGA
NPs (LF/Hep-PLGA NPs) [
206
]. These bioactive molecules were anchored together, as
well as separately, in order to evaluate their effects on IL-1
β
-treated tenocytes
in vitro
, and
in vivo
through a rat model of Achilles tendinitis [
206
]. Independent from the study condi-
tions (
in vitro
and
in vivo
), the nanoparticles containing lactoferrin (LF-PLGA NPs) and
LF/Hep-PLGA NPs managed to decrease the mRNA levels of the pro-inflammatory factors
COX-2, IL-1
β
, MMP-3, MMP-13, IL-6, and TNF-
α
, while increased the levels of the anti-
inflammatory cytokines IL-4 and IL-10 [
206
]. Another commonly used anti-inflammatory
drug is Diclophenac Diethylammonium. By fixing this medicine into gold nanoparticles
(GNPs), rats with a tendinous injury model were treated using a pulse therapeutic ultra-
sound technique, in which the drug was transported transcutaneously [
207
]. A significant
decrease in the pro-inflammatory cytokines IL-1
β
and TNF-
α
occurred in tendons with the
phonophoresis + diclophenac + GNPs treatment, and these nanoparticles showed to be
effective in transporting molecules to active inflammation sites [207].
Apart from the treatments with nanoparticles alone, a study conducted by Ciar-
dulli et al. addressed the use of NPs in scaffolds to study the immunomodulatory properties
of this combination. They developed a hyaluronate elastic band merged with a fibrin hy-
drogel scaffold (HY-FIB) containing hBMSCs and PLGA nano-carriers (PLGA-NCs) loaded
with human Growth Differentiation Factor 5 (hGDF-5) which has been shown to induce
the expression of genes linked to the neo-tendon phenotype [
139
,
208
]. This construct was
studied either in static or dynamic conditions in a 3D
in vitro
model to understand under
which settings exist the better tendon phenotype, as well as a pro-healing cytokine profile.
The results demonstrated that in dynamic conditions, pro-inflammatory cytokines IL-6,
TNF, IL-12A, and IL-1
β
displayed a less pronounced upregulation, while anti-inflammatory
TGF-β1 and IL-10 showed an increase by day 11 [139].
Altogether, bearing in mind the immunomodulatory possibilities offered by nanopar-
ticles/microspheres and electrospun scaffolds, more preliminary experiments using these
approaches in combination could be performed, hence allowing the obtention of a potential
synergic effect in modulating the immune system together with a different cellcompartment
within a tendon-like environment. Under these terms, Tomás et al. functionalized aligned

Cells 2022,11, 266 24 of 40
electrospun fiber threads of PCL matrix filled with iron oxide magnetic nanoparticles
(MNPs) attached to cellulose nanocrystals (MNP-CNCs) [
33
]. This 3D scaffold was seeded
with hASCs and activated with a magneto-mechanical stimulation. After 11 days, cells
cultivated on PCL/DT-NP5 under both static and magnetic conditions presented increased
expression of tendon-related markers. Furthermore, similar to the tenogenic differentia-
tion, the magnetic stimulation showed a higher immunomodulatory potential, increasing
the expression of anti-inflammatory cytokines IL-4 and IL-10, while downregulating the
pro-inflammatory cytokines IL-6 and COX-2 [33].
Considering the role of bioactive molecules together with the influence of the scaffolds’
intrinsic and extrinsic characteristics in immunomodulating the response of different cell
types both
in vitro
and
in vivo
, it is a promising idea for future experiments to study the
synergic effect that could have the electrospun scaffolds with aligned fibers engrafted
with these kinds of bioactive molecules on modulating the immune system response in
tendinopathies. Thus, the core concept in designing scaffolds, in combination with engi-
neered bioactive molecules and/or stem cells, resides in the modulation of the interaction
between the transplanted biomaterial-scaffold and the host tissue. This in turn allows a
pro-regenerative immune response, hence hindering fibrosis occurrence at the injury site
and guiding tendon regeneration.
5. Insights in the Molecular Pathways Regulating the Scaffold’s Mediated
Immunomodulation
As discussed in the previous paragraphs, the immune induction on stem and immune
cells is driven by several factors. Scaffold’s stiffness, porosity, surface hydrophilicity and
charge, and fibers’ alignment are all cells’ immunomodulator effectors (Figure 6). Indeed,
cells and tissues are sensitive to mechanical signals from their microenvironment, which
includes not only all components of force, stress, and strain, but also substrate rigidity,
topography, and adhesiveness [
209
]. Therefore, exploiting this cell’s mechanosensitiv-
ity, biomaterials and the resulting biophysical immunomodulatory cues could modulate
inflammatory pathways as well influence cells’ activity. This mechanism is called mechan-
otransduction and represents the ability of cells to react to mechanical cues transforming a
physical stimulus in a biological response, which is a critical component of musculoskeletal
tissue growth, homeostasis, healing, and degeneration [
209
–
212
]. The aforesaid envi-
ronmental changes can influence physiological mechanisms at the genetic, cellular, and
systemic levels [
213
]. Speaking of which, the understanding of the mechanosensitivity of
tissues and cells represents a promising direction to improve tendon TE.
Indeed, the tendon is a mechanosensitive tissue; this sensitivity, for instance, permits
mechanical loading-based therapies. Cells in the tendon are responsible for this adaptive
response. For example, the responsiveness of tendon fibroblasts to mechanical loading
has been well studied both
in vitro
and
in vivo
[
210
–
212
,
214
–
224
]. This has been employed
in TE, and several studies have demonstrated that the application of mechanical stimuli
on tendon biomimetic scaffolds engineered with stem cells leads to their differentiation
towards the tenogenic lineage in a more effective way compared to cells cultured un-
der static conditions [
225
]. Moreover, mechanical stimuli represent not only a booster of
tenogenic differentiation, but they also affect macrophages’ behavior. In this sense, Schoe-
nenberger et al. [
93
] explored macrophage activation and human macrophage–human
tendon fibroblasts’ (hTFs) crosstalk through the cooperative action of intrinsic topological
cues from PCL scaffolds (random or aligned fibers) and extrinsic mechanical stimuli. The
authors conducted
in vitro
and
in vivo
experiments using aligned or randomly oriented
PCL constructs in both mechanically loaded and unloaded conditions. The random fiber
topography promoted a pro-inflammatory behavior in macrophages and hTFs compared
to aligned fibers. Moreover, extrinsic mechanical loading was found to strongly reduce
macrophages’ pro-inflammatory markers both
in vitro
and
in vivo
. Even more, Schoenen-
berger et al. found that mechanical co-culture of macrophages and hTFs resulted in a

Cells 2022,11, 266 25 of 40
decrease in the CCR7 M1-marker, and increased mechanical loading resulted in an increase
in the M2 macrophage phenotype [93].
Cells 2022, 11, x FOR PEER REVIEW 25 of 41
mechanosensitivity, biomaterials and the resulting biophysical immunomodulatory cues
could modulate inflammatory pathways as well influence cells’ activity. This mechanism
is called mechanotransduction and represents the ability of cells to react to mechanical
cues transforming a physical stimulus in a biological response, which is a critical compo-
nent of musculoskeletal tissue growth, homeostasis, healing, and degeneration [209–212].
The aforesaid environmental changes can influence physiological mechanisms at the ge-
netic, cellular, and systemic levels [213]. Speaking of which, the understanding of the
mechanosensitivity of tissues and cells represents a promising direction to improve ten-
don TE.
Figure 6. The effect of mechanotransduction cues on RhoA pathway, which in turn act on YAP/TAZ
cellular distribution, and their influence on stem and immune cell immunomodulation.
Indeed, the tendon is a mechanosensitive tissue; this sensitivity, for instance, permits
mechanical loading-based therapies. Cells in the tendon are responsible for this adaptive
response. For example, the responsiveness of tendon fibroblasts to mechanical loading has
been well studied both in vitro and in vivo [210–212,214–224]. This has been employed in
TE, and several studies have demonstrated that the application of mechanical stimuli on
tendon biomimetic scaffolds engineered with stem cells leads to their differentiation to-
wards the tenogenic lineage in a more effective way compared to cells cultured under
Figure 6.
The effect of mechanotransduction cues on RhoA pathway, which in turn act on YAP/TAZ
cellular distribution, and their influence on stem and immune cell immunomodulation.
Therefore, the results of Schoenenberger et al. revealed that macrophage polarization
in response to biophysical cues is context based. Moreover, mechanosensitive response
of macrophages to biophysical signals generally overshadowed that of tendon fibroblasts,
with leading effects of crosstalk between these cell types observed in mechanical co-culture
models. Thus, these findings have highlighted a probable role for macrophages as key
mechano-sensitive cells that modulate tendon healing, and provide insights into how
biological response might be therapeutically modulated by rational biomaterial designs
that address the biomechanical niche of recruited cells [
93
]. However, for improved ten-
don regeneration, load must be applied with caution [
211
]. In general, there must be a
delicate balance struck between under stimulating and overloading the healing tendon-
to-bone interface. Several studies showed that removing all load from the healing site
is dangerous [
226
,
227
], but unnecessary load is also harmful [
228
]. This discovery may
also apply to TE mechanical loading applications [
211
]. During dynamic loading of the

Cells 2022,11, 266 26 of 40
tissue in culture, an even distribution of force throughout the entire sample is critical,
otherwise tissue integrity is compromised by overloading the mechanical connection re-
gions and/or by inhomogeneous mechanical stimulation [
225
]. Moreover, as demonstrated
by Ballotta et al., an overloading of macrophages seeded on PCL electrospun scaffolds
caused their polarization to pro-inflammatory M1 phenotype instead of pro-regenerative
M2 phenotype [229].
However, how are these mechanical cues converted into cascades of cellular and
molecular events that are able to instruct cells’ biology and enhance their immunomodula-
tion? To better understand the mechanotransduction mechanisms, the cellular components
that are involved in the transduction of mechanical forces are briefly reviewed.
The cellular membrane, which comes into direct contact with the ECM, is the primary
site of force transmission to the cell [
209
]. Mechanical deformations in the ECM can be
transmitted to the actin cytoskeleton which controls cell shape and cell motility and is
involved in several cellular functions. The cytoskeleton is composed of microfilaments,
microtubules, and intermediate filaments. Microfilaments are actin polymers that bind
virtually all the intracellular structures in a continuous, dynamic manner [
212
]. The ECM’s
forces on a cell are in equilibrium with the cell’s forces, and these forces are transmitted
through focal adhesion (FA) sites, integrins, cellular junctions, and the ECM. Changes in the
cytoskeleton caused by mechanical forces will set off complex signal transduction cascades
within the cell by activating integrins and stimulating G protein receptors, receptor tyrosine
kinases (RTKs), and MAPKs [
212
]. Integrins are transmembrane protein heterodimers
composed of
α
- and
β
-subunits and with three domains: ECM, single transmembrane,
and a cytoplasmic domain. The integrin’s ECM domain binds to substrates, while its
cytoplasmic domain connects various intracellular proteins that include the cytoskeleton
and multiple kinases, including focal adhesion kinase (FAK). Mechanical forces induce
integrin conformational activity in cells and enhance cell binding to the ECM. G proteins
are made up of a, b, and g subunits that pair membrane receptors and initiate intracellular
signaling cascades [
212
]. The g subunit of heterodimeric G proteins has been detected at
integrin-rich focal adhesion sites and adjacent to F-actin stress fibers (SFs), packs of actin
filaments and non-muscle myosin II, as well as in other crosslinking proteins [
212
,
230
,
231
].
RTKs are a kind of cell membrane protein that are phosphorylated when stretched or
sheared. The MAPK is a protein that travels into the nucleus and interacts with transcription
factors and promoters to change gene expression, as well as the ribosomal S6 kinase (RSK)
to initiate translation [212,232].
In this brief review of the cellular components involved in the transduction of mechani-
cal forces, FA have a central role. Cells in culture form FA, which are sites of tight adhesions
to the underlying ECM. FA are multi-protein integrin-containing structures that cross the
plasma membrane and serve as a structural link between the actin cytoskeleton and the
ECM, as well as signal transduction regions involved in growth regulation [
233
]. In detail,
FA are formed by the assembly of transmembrane proteins (integrins) that interact with
ECM components, such as fibronectin, vitronectin, collagens, and laminins. The extracellu-
lar subdomains of the integrin subunits contact the ECM, while the cytoplasmic tail interacts
with cytoskeletal actin through several docking proteins such as vasodilator-stimulated
phosphoprotein (VASP), paxillin, tyrosin kinase Src and FAK [
209
,
231
]. FAK is one of the
first molecules recruited to form FA in response to external mechanical stimuli [
209
]. Its
activation by autophosphorylation is considered as the trigger for intracellular mechan-
otransduction, as it activates downstream mechanotransducers in the cytoplasm. Moreover,
the activation of FAK can result in increased cell proliferation through the activation of the
MAPK family member extracellular signal-regulated kinases (ERKs) [
231
]. Cytoskeletal
contraction, cell spreading, and other downstream signals reinforce FAK activation in
a positive loop, so exogenous force can increase FAK phosphorylation. Moreover, this
interaction between FAK and the contractile cytoskeletal network is controlled in the cell in
order to maintain tension at key cell sites and manage force transfer to the nucleus [209].

Cells 2022,11, 266 27 of 40
FA are dynamic structures that expand and contract in size because of protein recruit-
ment and disassembly in response to mechanical forces [
231
]. Their assembly is regulated
by the GTP-binding protein Rho [
233
]. Rho GTPases are small GTPases, members of the
Ras superfamily, that work as molecular switches by binding to guanosine triphosphate
(GTP) and guanosine diphosphate (GDP). Many cellular processes have been stated to be
controlled by them, including actin cytoskeleton remodeling, transcription, cell growth
and proliferation, cell motility, morphology, and cell cycle progression. So far, more than
20 members of the Rho GTPase family have been discovered. RhoA, Cdc42, and Rac1 have
received the most attention [231].
Tension and myosin are two significant contributors in the development and matura-
tion of SFs and FA. New focal complexes form at the lamellipodia (a cytoskeletal protein
actin projection) and are primarily regulated by Rac1 and Cdc42. Mechanical force facilitates
the maturation of emerging focal complexes into focal adhesions by recruiting additional
proteins and promoting actin polymerization. Previous research has shown that RhoA
activity is needed for the formation of SF and FA [
231
,
234
]. Moreover, by using myosin
inhibitors it has been shown that inhibiting RhoA-mediated myosin activity resulted in a
failure to form SFs and FA [
231
,
235
]. Current knowledge suggests that mechanical tension
activates RhoA signaling pathways and also exposes the binding sites in the mechanosen-
sors [
231
]. Activated RhoA promotes contractility, which results in isometric tension in cells
that are tightly adhered to the substrate [
233
]. In detail, RhoA activate ROCK, a RhoA effec-
tor, which in turn phosphorylates myosin II, which feeds back positively to increase cellular
tension [
231
]. Moreover, ROCK causes actin filament bundling and integrin aggregation in
the membrane plane. This last element activates the FAK, resulting in the formation of a
multicomponent signaling complex [
233
]. Some mechanosensor proteins, such as talin, can
also undergo conformational changes because of increased stress. Talin stretching exposes
new binding sites for the recruitment of other focal adhesion proteins such as vinculin, one
of the key components of FA inner core [
209
,
231
]. The stretch-sensitive adaptor protein
p130Cas is another mechanosensor that contains SH3 domains by which it interacts with
vinculin and FAK at the FA site. When mechanically stretched, p130Cas exposes buried
tyrosine residues that can be phosphorylated by Src kinase [209,231].
Topography has demonstrated to influence the arrangement of integrins and the for-
mation of FA [
231
]. Because of the nanometer-sized range of integrins, cells can distinguish
topographic changes down to the nanometer scale. During initial adhesion to the microen-
vironment, cells probe and migrate along the surface using membrane protrusions such as
lamellipodia and filopodia as contact guidance. Topographical cues generate mechanical
forces that are transmitted into the nucleus via integrins that are linked to the cytoskeleton,
according to growing evidence. Topographical cues, on the other hand, may produce
mechanical forces that are exerted by FA, activating focal adhesion signaling pathways
through FAK and focal adhesion-associated proteins [
231
]. As previously seen, stiffness
represents a topographic feature of scaffold, able to module cells’ immune propriety. Stiff-
ness of the substrate influences integrin clustering, as well as FA assembly and turnover.
Cells grown on stiff substrates exhibit elevated intracellular tension, which is distinguished
by the presence of stress fibers. The cell-generated contractile force is fought by the stiffness
of the ECM, resulting in increased force at the cell–matrix interface that further enhances FA
assembly. Consequently, cells grown on stiffer substrates have more focal adhesions [
231
].
Patel et al., investigated the role of mechanosensitivity in macrophage function, finding
that macrophage elasticity (elastic modulus), which is mediated by substrate stiffness, is
actively dependent on actin polymerization and RhoGTPase activity. On stiff (150 kPa)
polyacrylamide substrates, RAW 264.7 cells (monocyte/macrophage-like cells derived from
an Abelson leukemia virus-transformed BALB/c mouse cell line) exhibited organized actin
filaments and filopodial projections. However, when treated with a Rho-GTPase inhibitor
(C. Difficile toxin), cells resembled those on softer (1.2 kPa) substrates, with an absence
of organized actin fibers in projections. Moreover, the elasticity and phagocytic capacity
of cells cultured on stiff substrates were also significantly higher, implying that substrate

Cells 2022,11, 266 28 of 40
elasticity modulates macrophage elasticity and phagocytosis through actin polymeriza-
tion [
236
]. Moreover, using mouse BMM
Φ
s, Beningo and Wang investigated the effects of
substrate stiffness on macrophage function. They found that stiff polyacrylamide particles
were phagocytized preferentially over soft polyacrylamide particles of equal chemistry via
a Rac-1 mediated mechanosensory pathway [237].
In response to mechanical perturbations, mechanical homeostasis in cells is maintained
by modifying focal adhesion ligand affinity and by regulating focal adhesion assembly and
disassembly. Therefore, mechanical stimuli applied to stem cell engineered scaffold
in vitro
could cause an upregulation of focal adhesion components [
238
]. Then, the mechanical
information is propagated at the cytoskeleton level, where it affects proteins residing at
the membrane or in the cytoplasm, causing structural modification and their shuttling to
the nucleus [
209
]. One of the mediators between the mechanical stimuli sensed by the
cytoskeleton and the associated cell response are Yorkie-homologues YAP (Yes-associated
protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as
WWTR1), transcriptional co-activators being the downstream effectors of Hippo path-
way [
33
,
239
,
240
]. The activity of YAP and TAZ is important for the growth of entire organs,
the amplification of tissue-specific progenitor cells during tissue renewal and regeneration,
and cell proliferation. Mechanical signals represent a second aspect of the YAP/TAZ func-
tion [
241
]. The mechanotransduction role of YAP is related to its ability to promote the
transcription of genes involved in cell–matrix interaction, ECM composition, and cytoskele-
ton integrity [
209
]. YAP/TAZ represent a new class of shuttling proteins that function as
mechanotransducers by going back and forth from the nucleus [
209
]. Since activation of
YAP and TAZ results in their accumulation in the nucleus, a significant layer of control of
YAP and TAZ occurs at the level of their subcellular distribution. YAP/TAZ are mostly
cytoplasmic, but when activated, they shuttle from the cytoplasm to the nucleus to regulate
gene expression [
84
,
242
,
243
]. Cell structure, the rigidity and topology of the ECM substrate,
and shear stress all influence this cellular distribution. YAP and TAZ, for example, are
found in the cytoplasm of cells with low levels of mechanical signaling, such as rounded
cells connected to a soft ECM. On the other hand, they are nuclear in cells that experience
elevated levels of mechanical signaling, such as cells cultured on stiff substrates or cells that
experience deformation and cytoskeletal stress [
239
,
242
,
244
,
245
]. The mechanosensitive
role of YAP and TAZ also results from their interaction with the mechanosensory systems
described previously, such as integrins, adaptor proteins such as vinculin and talins, FAK,
and SRC-family kinases. For example, in some cell types, inhibitors of non-muscle myosin
II or ROCK can direct YAP activation by a stiff ECM. Furthermore, Rho signaling is needed
for YAP and TAZ action, which has been experimentally exploited in a variety of systems,
either genetically or using Rho inhibitors [
239
,
242
,
244
–
247
]. Then, YAP and TAZ serve as
nuclear relays for mechanical signals induced by ECM rigidity and cell structure. Indeed,
for example ECM stiffness or cell geometry influence their activity. This control includes
Rho GTPase activity and actomyosin cytoskeleton stress but is not contingent on the Hippo
cascade, it is a parallel and independent pathway (Figure 6) [239].
Moreover, YAP and TAZ were founded as binding proteins for Smads, a key transducer
of the TGF-
β
[
159
,
248
–
250
]. Cytoplasmic YAP/TAZ interact with SMAD2/3 through the
TAZ coiled-coil domain and participate in Smad2/3 cytoplasmic retaining, even overruling
the effects of high levels of TGF-
β
ligands [
249
,
250
]. Moreover, YAP binds SMAD7, enhanc-
ing its inhibitory activity against the TGF-
β
receptors [
241
]. SMAD2/3 were investigated
together with ERK1/2 pathways by Liu et al., Indeed, they explored the adhesion and
proliferation of rabbit tenocytes and fibroblasts on multi-layered electrospun PCL–amnion
nanofibrous membranes. Liu et al., scaffolds resulted in ERK1/2 and SMAD2/3 phospho-
rylation upregulation, adhesion and proliferation of tenocytes, fibroblast promotion, and
collagen synthesis increase [251].
Quite recently, Tomás et al., investigated the magneto-mechanical stimulation of
hASCs seeded on an electrospun PCL fibrous aligned scaffold functionalized with hybrids
of cellulose nanocrystals decorated with magnetic nanoparticles. Thus, they evaluated

Cells 2022,11, 266 29 of 40
the expression of YAP/TAZ in hASCs cultured for both 11 and 21 days under magneto-
mechanical stimulation or in static conditions. Immunofluorescence images of hASCs
cultured for 11 days under both conditions showed a predominant YAP/TAZ nuclear
expression. The ability of the scaffolds to cause YAP/TAZ activation under both conditions
shows that their aligned topography resulted in sufficient cell cytoskeleton stimulation.
However, the nuclear to cytoplasmatic YAP/TAZ ratio in stimulated cells at day 11 were
significantly higher than the static culture condition. On the other hand, immunofluores-
cence images of hASCs cultured for 21 days revealed a predominance of cytoplasmatic
YAP/TAZ, most likely due to cell crowding and a decline in proliferation [
33
]. The findings
of Tomás et al. match perfectly with the reports of Wan et al. Indeed, they found that
both FAK and YAP/TAZ signaling are necessary mechanotransduction pathways through
which aligned fibers stimulate the immunomodulatory role of ASCs, whose paracrine
secretions can induce M2 phenotypic changes in macrophages [
84
]. In detail, they selected
a PLLA electrospun fibrous scaffold with two different orientations (random vs. aligned) to
investigate the effects of fiber orientation on the secretory immunomodulatory behavior of
human ASCs. Then, Wan et al. used the conditioned media from ASCs cultured on aligned
fiber (for 48 h) to cultivate human ASCs. To illustrate the involvement of FAK, ERK1/2, and
YAP/TAZ in this modulation, small molecular inhibitors, including PF573228, PD98059,
and Verteporfin, were added to the medium for 48 h prior to further characterization. ASCs
cultured on aligned fibrous matrices secreted a significantly higher number of immunomod-
ulatory factors (COX-2, TSG-6, HGF, TGF-
β
, MHC-G, IL-1ra, M-CSF and MCP-1) compared
to that of ASCs cultured on random fibrous matrices. Moreover, these secreted factors by
ASCs on aligned fibrous scaffolds addressed macrophages to an anti-inflammatory M2
phenotype as noticeable by the reduced secretion of pro-inflammatory factors (e.g., IL-1b)
and enhanced expression of M2 surface markers (CD163 and CD206). Interestingly, ASCs
on aligned fibers expressed a preferential nuclear YAP/TAZ localization (43%) to those
on random fibers (9%). On the other hand, elevated cytoplasmic YAP/TAZ staining was
observed in ASCs on random fibers (28%) compared to those on aligned fibers (2%). More-
over, Wan et al. found that the enhanced immunomodulatory functions of ASCs on aligned
fibrous matrices was stopped by treatment with inhibitors of FAK (PF573228), ERK1/2
(PD98059), and YAP/TAZ (Verteporfin), suggesting that FAK-ERK1/2 and YAP/TAZ sig-
naling are involved in mediating the fiber orientation-induced immunomodulation changes
in ASCs (Figure 6) [84].
Thus, mechanotransduction could represent a possible explanation for all the different
mechanisms through which the intrinsic and extrinsic properties of the scaffolds can
immunomodulate stem and immune cells. Indeed, the understanding of the involved
mechanisms could lead to the development of novel immunoengineering strategies to be
applied in tendon TE.
6. Conclusions
Immunoengineering has been introduced as a key challenge discipline in TE with the
aim to immunomodulate the inflammatory response using scaffolds. Indeed, understand-
ing the crosstalk amongst scaffold, stem and immune cells might greatly modulate the
immune response through the design of immunoinformed scaffolds, which in turn could
improve the regenerative potential of immune and stem cells, hence resolving the inflam-
matory response and avoiding the formation of fibrotic scar tissue. This can be achieved
by stimulating macrophage polarization towards an anti-inflammatory phenotype and by
recruiting stem cells within the damage tissue improving hence their immunomodulatory
properties. Indeed, controlling the intrinsic characteristics of electrospun scaffolds in terms
of fiber topography, fiber diameter, pore size and surface chemistry together with the
extrinsic ones including mechanical stimuli and scaffold degradation behavior has offered
new insights to improve the scaffolds’ regenerative potential to deal with tendinopathies.
It has been demonstrated that fiber alignment with adequate fiber diameter size, surface
chemistry and functionalization of electrospun scaffolds with specific bioactive molecules

Cells 2022,11, 266 30 of 40
can greatly increase stem and immune cell recruitment and switch their immune response
towards a pro-regenerative response. Studies concerning the immunoregenerative strate-
gies applied to tendon TE are still few, as confirmed by the scientometric analysis. Further
studies are required to better understand the mechanisms behind tendon regeneration as
well as scaffold–immune system interaction, in order to improve the performance of the
electrospun scaffolds, hence allowing the development of new immunoinformed scaffolds
able to completely regenerate tendon damages.
Author Contributions:
M.E.K. coordinated the review and wrote the paragraphs: Introduction,
Biomimetic scaffold applied for tendon TE, Immune response induced by scaffold implantation, Scaf-
fold immunoregenerative strategies for tendon TE applications. G.P. wrote the following paragraphs:
Immuno-induction of scaffold on immune cells, Insight in the molecular pathways regulating the
scaffold’s mediated immunomodulation, and the Conclusions. A.C.V., M.F. and A.A.H.M. wrote
the paragraph: Biological strategies to enhance the immunoregenerative potential of the scaffolds.
M.R.C. and A.M. wrote the paragraph immuno-induction of scaffold on stem cells. L.L. and A.R.B.
supervised and revised the paragraphs: Biomimetic scaffold applied for tendon TE and scaffold
immunoregenerative strategies for tendon TE applications. V.R. supervised the whole review and
critically reviewed together with B.B. the draft manuscript. A.M. and P.B. supervised the paragraphs:
Immuno-induction of scaffold on stem cells and biological strategies to enhance the immunoregen-
erative potential of the scaffolds. V.R., A.M., P.B. and B.B. were involved in the conceptualization
of the review. V.R. and B.B. provided funding. M.E.K., G.P., O.D.G. and M.T. elaborated the figures.
V.R., M.R., F.S., F.B., L.L. and A.R.B. critically reviewed the final version of the review. All authors
validated the data and reviewed the manuscript. All authors have read and agreed to the published
version of the manuscript.
Funding:
This research was funded by Perspective for Future Innovation in Tendon Repair H2020-
MSCA-ITN-EJD-P4 FIT—Grant Agreement ID: 955685.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
The figures were obtained using www.BioRender.com (accessed on 31 May 2021).
Conflicts of Interest: The authors declare no conflict of interest.
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