ArticlePDF AvailableLiterature Review

Promoting Tissue Regeneration by Modulating the Immune System

January 2017
Acta Biomaterialia 53(177)

January 2017
53(177)

DOI:10.1016/j.actbio.2017.01.056

License
CC BY-NC-ND 4.0

Authors:

Ziad Julier

Monash University (Australia)

Anthony Park

Thermo Fisher Scientific

Priscilla S Briquez

University of Chicago

Mikaël M Martino

Monash University; Osaka University

Statement of significance: Most regenerative strategies have not yet proven to be safe or reasonably efficient in the clinic. In addition to stem cells and growth factors, the immune system plays a crucial role in the tissue healing process. Here, we propose that controlling the immune-mediated mechanisms of tissue repair and regeneration may support existing regenerative strategies or could be an alternative to using stem cells and growth factors. The first part of this review we highlight key immune mechanisms involved in the tissue healing process and marks them as potential target for designing regenerative strategies. In the second part, we discuss various approaches using biomaterials and drug delivery systems that aim at modulating the components of the immune system to promote tissue regeneration.

Apparent inverse relationship between regenerative and immune capacities during evolution or development. Lower vertebrates such as fishes and amphibians have the ability to completely regenerate many of their tissues. In mammals, regenerative capacities depend on the developmental stage (i.e. fetal, neonatal, and adult). Immune competences have increased during evolution and also increase with life stage in mammals.

…

The main actors of the immune response following tissue injury. (A) Kinetic of immune cell mobilization after tissue injury. Tissue resident cells including tissueresident macrophages and cdT cells sense tissue damage and trigger the mobilization of other immune cells. Neutrophils are followed by monocytes/macrophages and T cells.

…

Strategies based on biomaterials and drug delivery systems to promote tissue regeneration by controlling the immune system. Biomaterial-based strategies aiming at improving the healing process through immunomodulation can be achieved either by the biomaterial itself and/or by the delivery of immunomodulators. Strategies most commonly aim either at the delivery of pro-inflammatory modulators to initiate the healing process or the delivery of anti-inflammatory modulators to promote the resolution phase via anti-inflammatory/anti-fibrotic macrophages. More complex strategies rely on a sequential delivery of pro-inflammatory and anti-inflammatory molecules to exert a more comprehensive control over the tissue healing process. Black arrows indicate a differentiation path. Red arrows indicate induction. Blue arrows indicate inhibition. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

…

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Review article

Promoting tissue regeneration by modulating the immune system

Ziad Julier

a,1

, Anthony J. Park

a,1

, Priscilla S. Briquez

, Mikaël M. Martino

⇑

European Molecular Biology Laboratory Australia, Australian Regenerative Medicine Institute, Monash University, Victoria 3800, Australia

Institute for Molecular Engineering, University of Chicago, Chicago, IL 60637, USA

article info

Article history:

Received 31 October 2016

Received in revised form 3 January 2017

Accepted 20 January 2017

Available online 22 January 2017

Keywords:

Regenerative medicine

Immune system

Biomaterials

Drug delivery systems

Cytokines

Inﬂammation

Scarring

Fibrosis

Macrophages

T cells

abstract

The immune system plays a central role in tissue repair and regeneration. Indeed, the immune response

to tissue injury is crucial in determining the speed and the outcome of the healing process, including the

extent of scarring and the restoration of organ function. Therefore, controlling immune components via

biomaterials and drug delivery systems is becoming an attractive approach in regenerative medicine,

since therapies based on stem cells and growth factors have not yet proven to be broadly effective in

the clinic. To integrate the immune system into regenerative strategies, one of the ﬁrst challenges is to

understand the precise functions of the different immune components during the tissue healing process.

While remarkable progress has been made, the immune mechanisms involved are still elusive, and there

is indication for both negative and positive roles depending on the tissue type or organ and life stage. It is

well recognized that the innate immune response comprising danger signals, neutrophils and macro-

phages modulates tissue healing. In addition, it is becoming evident that the adaptive immune response,

in particular T cell subset activities, plays a critical role. In this review, we ﬁrst present an overview of the

basic immune mechanisms involved in tissue repair and regeneration. Then, we highlight various

approaches based on biomaterials and drug delivery systems that aim at modulating these mechanisms

to limit ﬁbrosis and promote regeneration. We propose that the next generation of regenerative therapies

may evolve from typical biomaterial-, stem cell-, or growth factor-centric approaches to an immune-

centric approach.

Statement of Signiﬁcance

Most regenerative strategies have not yet proven to be safe or reasonably efﬁcient in the clinic. In addi-

tion to stem cells and growth factors, the immune system plays a crucial role in the tissue healing pro-

cess. Here, we propose that controlling the immune-mediated mechanisms of tissue repair and

regeneration may support existing regenerative strategies or could be an alternative to using stem cells

and growth factors. The ﬁrst part of this review we highlight key immune mechanisms involved in the

tissue healing process and marks them as potential target for designing regenerative strategies. In the

second part, we discuss various approaches using biomaterials and drug delivery systems that aim at

modulating the components of the immune system to promote tissue regeneration.

Ó2017 Acta Materialia Inc. Published by Elsevier Ltd. This is an open access article underthe CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction . . . ....................................................................................................... 14

2. The main actors of the immune response following tissue injury . . . . . . . . . . . . . . . . . .............................................. 14

2.1. Danger signals . . . . . . . . . .......................................................................................... 15

2.2. Neutrophils and mast cells . . . . . . . . . . . . . . . .......................................................................... 16

2.3. Monocytes and macrophages . . . . . . . . . . . . . ................................................... ....................... 16

2.4. Pericytes. . . . . . . . . . . . . . .......................................................................................... 17

http://dx.doi.org/10.1016/j.actbio.2017.01.056

1742-7061/Ó2017 Acta Materialia Inc. Published by Elsevier Ltd.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

⇑

Corresponding author at: Australian Regenerative Medicine Institute, 15 Innovation Walk, Building 75, Level 1, Martino lab, Victoria 3800, Australia.

E-mail address: mikael.martino@monash.edu (M.M. Martino).

These authors contributed equally.

Acta Biomaterialia 53 (2017) 13–28

Contents lists available at ScienceDirect

Acta Biomaterialia

journal homepage: www.elsevier.com/locate/actabiomat

2.5. Dendritic cells . . . ................................................................................................ 18

2.6. T cells. . . . . . . . . . ................................................................................................ 18

2.7. B-cells. . . . . . . . . . ................................................................................................ 18

3. Promoting tissue regeneration by modulating the immune system . . . . . . . . . . .................................................... 18

3.1. Immune modulation by the physicochemical properties of biomaterials . . . . . . . . . . .......................................... 19

3.2. Immune modulation by decellularized ECM . . . . . . . . . . . . . . ............................................................. 20

3.3. Delivery of inflammatory molecules . . . . ............................................................................. 20

3.3.1. SDF-1. . . . . . . . . . . . . . . . . .................................................................................. 20

3.3.2. PGE2 . . . . . . . . . . . . . . . . . .................................................................................. 20

3.4. Delivery of anti-inflammatory molecules ............................................................................. 21

3.4.1. Pro-resolving mediators . . .................................................................................. 21

3.4.2. Inhibitors of TNF-

........................................................................................ 21

3.4.3. Inhibitors of the NF-

B pathway. . . . . . . . . . . .................................................................. 21

3.4.4. Anti-inflammatory cytokines . . . . . . . . . . . . . . .................................................................. 22

3.4.5. siRNA. . . . . . . . . . . . . . . . . .................................................................................. 22

3.4.6. miRNA . . . . . . . . . . . . . . . . .................................................................................. 22

3.5. Extracellular vesicles. . . . . . . . . . . . . . . . . ............................................................................. 22

3.6. Codelivery of immune modulators and morphogens . . . . . . . ............................................................. 23

4. Conclusion and future perspectives . . . . . . . . . . . . . . . . ....................................................................... 23

Acknowledgements . . . . . . . . . . .......................................................................................... 24

References . .......................................................................................................... 24

1. Introduction

While remarkable progress has been achieved in understanding

the cellular and molecular mechanisms of tissue repair and regen-

eration, it remains unexplained why mammals have a tendency for

imperfect healing and scarring rather than regeneration. There is

ample evidence in different model organisms indicating that the

immune system is crucial to determine the quality of the repair

response, including the extent of scarring, and the restoration of

organ structure and function. A widespread idea derived from ﬁnd-

ings in diverse species is that the loss of regenerative capacity is

linked to the evolution of immune competence (Fig. 1). Still, there

are many situations where the immune response to tissue injury

promotes tissue healing. Indeed, the relationship between tissue

healing and the immune response is very complex, since there

are both negative and positive roles, depending on the tissue, organ

and life stage (embryonic, neonatal or adult) [1]. The type of

immune response, its duration and the cells involved can drasti-

cally change the outcome of the tissue healing process from incom-

plete healing and repair (i.e. scarring or ﬁbrosis) to complete

restoration (i.e. regeneration).

In regenerative medicine, strategies based on stem cells and

growth factors have not yet proven broadly effective in the clinic.

Here, we propose that immune-mediated mechanisms of tissue

repair and regeneration may support existing regenerative strate-

gies or could be an alternative to using stem cells and growth fac-

tors. In the ﬁrst part of this review, we present key immune

mechanisms involved in the tissue healing process, in order to

highlight potential targets. In the second part, we discuss various

approaches using biomaterials and drug delivery systems that

aim at modulating the components of the immune system to pro-

mote tissue repair and regeneration.

2. The main actors of the immune response following tissue

injury

An immune response almost always follows tissue damage and

this response is usually resolved within days to weeks after an

injury. The ﬁrst phase of the immune response involves compo-

nents of the innate immune system, which provide instant defense

against potential pathogens invading the damaged tissue. How-

ever, even in the absence of pathogens, the immune response ini-

tially triggered by danger signals released from damaged tissues

produces a so-called sterile inﬂammation [2,3]. In many if not all

tissues, the innate immune response strongly modulates the heal-

ing process. For instance, macrophages and their various pheno-

types play a predominant role in the restoration of tissue

homeostasis by clearing away cellular debris, remodeling the

extracellular matrix (ECM), and synthesizing multiple cytokines

and growth factors. The innate immune response is then followed

by the activation of the adaptive immune system. Although this

was originally thought of as a secondary actor in the tissue healing

process, the adaptive immune response to tissue injury most likely

plays a critical role during tissue repair and regeneration, in partic-

ular the activity of T cells. While a large research effort has focused

on how transplanted mesenchymal stem cells (MSCs) modulate T

cell activities and immune tolerance [4,5], our understanding of

how T cells modulate tissue-resident stem cells and the tissue

healing process is just beginning. In the next sections, we review

the roles and importance of the main actors that shape the

immune response following tissue injury.

Regenerative capacity

Immune competence

Neonatal

Fetal

Adult

Fish

Amphibians

Reptiles

Birds

Mammals

Fig. 1. Apparent inverse relationship between regenerative and immune capacities

during evolution or development. Lower vertebrates such as ﬁshes and amphibians

have the ability to completely regenerate many of their tissues. In mammals,

regenerative capacities depend on the developmental stage (i.e. fetal, neonatal, and

adult). Immune competences have increased during evolution and also increase

with life stage in mammals.

14 Z. Julier et al. / Acta Biomaterialia 53 (2017) 13–28

2.1. Danger signals

Directly after tissue injury, a local inﬂammation is induced in

response to damage-associated molecular patterns (DAMPs, or

alarmins) and pathogen-associated molecular patterns. Endoge-

nous danger signals are typically released from necrotic or stressed

cells and damaged ECM [2,3]. Well-known DAMPs include heat

shock proteins (HSP), monosodium urate, high-mobility group

box protein 1 (HMGB1), extracellular ATP, and nucleic acids includ-

ing mitochondrial DNA. Inﬂammatory cytokines such as inter-

leukin (IL)-1

and IL-33 can also work as DAMPs and are

released passively from necrotic cells. In addition, fragments from

Fig. 2. The main actors of the immune response following tissue injury. (A) Kinetic of immune cell mobilization after tissue injury. Tissue resident cells including tissue-

resident macrophages and

dT cells sense tissue damage and trigger the mobilization of other immune cells. Neutrophils are followed by monocytes/macrophages and T cells.

The relative amount of each cell type recruited is not represented. (B) Overview of the initial inﬂammatory phase following tissue injury. Chronological events are represented

from left to right. Tissue damage is sensed by tissue-resident macrophages via DAMPs. Neutrophils are the ﬁrst circulating immune cells recruited to the site of injury,

promoting inﬂammation and monocyte/macrophage recruitment. The inﬂammation is initially maintained by pro-inﬂammatory M(IFN-

) macrophages, before being

eventually resolved with the help of M(IL-4) macrophages. (C) Overview of the immune mechanisms that can impair tissue healing or drive to scarring and ﬁbrosis. M(IFN-

)

macrophages stimulate effector T cells in a positive-feedback loop. Effector T cells may also inhibit the regenerative capacity of tissue resident stem/progenitor cells via

inﬂammatory cytokines. M(IL-4)-like macrophages with a pro-ﬁbrotic activity encourage ECM protein deposition and subsequent ﬁbrosis (scarring), preventing full

regeneration of the original tissue. Pericytes increase immune cell mobilization and differentiate into scar forming myoﬁbroblasts via growth factors such as TGF-b1. (D)

Overview of the pro-regenerative immune mechanisms. A critical amount of macrophages displaying an anti-inﬂammatory/anti-ﬁbrotic phenotype (e.g. M(IL-10)-like)

contribute to regeneration through a crosstalk with Tregs, which in turn help sustain the anti-inﬂammatory/anti-ﬁbrotic phenotype via secretion of anti-inﬂammatory

cytokines such as IL-10. Tregs may also enhance the regenerative capacity of endogenous stem/progenitor cells through secretion of growth factors. Th2 cells induce/maintain

anti-ﬁbrotic/anti-inﬂammatory macrophages. Black arrows indicate a differentiation path or secretion of immune modulators/morphogens. Black dashed arrows indicate a

hypothetical differentiation path. Red arrows indicate induction. Blue arrows indicate inhibition.

Z. Julier et al. / Acta Biomaterialia 53 (2017) 13–28 15

ECM components such as hyaluronic acid, collagen, elastin, ﬁbro-

nectin and laminin all stimulate inﬂammation [6,7].

Toll-like receptors (TLRs) and other types of pattern recognition

receptors recognize danger signals and trigger inﬂammation via

the activation of the transcription factors NF-

B or interferon-

regulatory factors. TLRs activate tissue-resident macrophages and

promote the expression of chemoattractants for neutrophils,

monocytes and macrophages (Fig. 2A, B). They also induce the

expression of pro-inﬂammatory cytokines such as tumor necrosis

factor-

(TNF-

), IL-1band IL-6 [8,9]. Interestingly, inﬂammation

in response to necrotic cells is mostly mediated by IL-1 receptor

(IL-1R), which leads to NF-

B activation [10]. IL-33 also acts as a

primary danger signal via the ST2 receptor [11]. However, the

dominant danger signal varies in the context of the injury, includ-

ing the location, magnitude, manner of cell death, and time point

after the injury [3].

TLRs and IL-1R1 have been shown to negatively inﬂuence the

repair of several tissues [12–22]. For instance, the harmful effect

of TLR4 signaling is apparent in many organs, as seen by the pro-

tection of TLR4-mutant or deﬁcient mice after hepatic, renal, car-

diac, and cerebral ischemia-reperfusion [12–16]. Similarly, IL-1R1

signaling critically regulates infarct healing [17] and disruption

of IL-1 signaling can improve the quality of wound healing

[18,20]. In addition, it has been shown that IL-1R1/MyD88 signal-

ing negatively regulates bone regeneration in the mouse by impair-

ing the regenerative capacities of mouse MSCs [23]. While TLRs

and IL-1R1 seem to be detrimental for many tissues, studies have

shown that skin wound healing is impaired in mice deﬁcient for

various TLRs [24–26]. For example, TLR4 signaling helps wound

healing through stimulation of transforming growth factor-b

(TGF-b) and CC chemokine ligands (CCL)-5 expression [24].

Another endogenous TLR4 agonist, the extra domain A type III

repeat of ﬁbronectin (FNIII EDA) [27], has been reported to be over-

expressed at sites of injury [28,29], and is known to inﬂuence skin

repair [30]. For instance, wound healing in FNIII EDA knockout

mice is abnormal [31].

Overall, it is clear that danger signals signiﬁcantly inﬂuence the

healing process at early stages. They are indeed necessary to

induce inﬂammation, mainly via NF-

B, and they are also involved

in neutrophil, monocyte and macrophage mobilization. Yet, in the

case of ischemia-reperfusion and bone regeneration TLR and IL-R1

signaling seem to be detrimental.

2.2. Neutrophils and mast cells

Neutrophils are usually the ﬁrst inﬂammatory cell recruited at a

site of injury, enhancing host defense and wound detection while

removing contaminants [32] (Fig. 2A, B). The recruitment of neu-

trophils requires changes on endothelium surface mediated by his-

tamine, cytokines, and chemokines such as C-X-C motif ligand

(CXCL) 8 that are released by tissue resident cells upon pattern

recognition receptor and TLR activation. This will triggers a recruit-

ment cascade involving the capture of free ﬂowing neutrophils, fol-

lowed by their transmigration from the vasculature to the tissue,

facilitated by an increase permeability of the blood vessels at the

injured site [32]. Neutrophils produce antimicrobial substances

and proteases that help kill and degrade potential pathogens

[33]. In addition, they secrete cytokines and growth factors such

as IL-17 and vascular endothelial growth factor (VEGF)-A, which

recruit and activate more neutrophils and other inﬂammatory

cells, promote angiogenesis, and stimulate proliferation of cells

such as ﬁbroblasts, epithelial cells and keratinocytes (Fig. 2B)

[32–34].

Neutrophils are also able to deploy neutrophil extracellular

traps (NETs) [35], made of chromatin, proteins and enzymes, able

to catch pathogens and either directly kill them or facilitate their

phagocytosis. Yet, the formation of NETs (or NETosis) needs to be

tightly regulated, since NETosis might impair the healing process.

For example, there are evidences of delayed reepithelization in

the case of diabetes where NETosis is enhanced [36]. This is consis-

tent with the observation that neutrophil depletion might acceler-

ate wound closure in diabetic mice [37].

Importantly, neutrophils exhibit anti-inﬂammatory capacities.

They facilitate the recruitment of monocytes and macrophages,

which phagocytize dying neutrophils and other cellular debris.

Thus, neutrophils promote their own removal and thereby con-

tribute to the resolution of inﬂammation (Fig. 2B) [32]. For exam-

ple, following myocardial infarction, neutrophils help controlling,

macrophages, polarization, which is a critical step for proper tissue

repair [38]. Therefore, tightly controlling neutrophil mobilization

and functions could be an interesting strategy to promote tissue

repair and regeneration. For instance, pro-resolving mediators

derived from omega 3 fatty acid have the ability to modulate neu-

trophil mobilization as well as their ingestion by macrophages

[39].

Similarly to neutrophils, mast cells participate in the innate

immune response by secreting an array of effector molecules to

recruit eosinophils and monocytes. A large number of mast cells

seem to be detrimental for tissue regeneration. For example, they

enhance acute inﬂammation and promote scarring in the central

nervous system [40]. Moreover, they persist at high numbers in

chronic wounds [41]. Nevertheless, controlling mast cells to pro-

mote regeneration rather than repair and scarring should be tem-

pered, since mast cells also produce anti-inﬂammatory mediators,

suggesting alternative and dynamic functions for these cells during

repair [40].

2.3. Monocytes and macrophages

In addition to their role as scavenger cells that phagocytise cel-

lular debris, invading organisms, neutrophils and other apoptotic

cells, macrophages actively regulate the tissue healing process

[42]. A population of tissue macrophages resides in most tissues,

but a large number of macrophages are recruited after tissue

injury, and these often greatly exceed the population of tissue-

resident macrophages [43]. The recruited and resident populations

proliferate and undergo marked phenotypic and functional

changes, in response to the tissue microenvironment. Importantly,

macrophages are a source of various proteases, cytokines, growth

factors, ECM components and soluble mediators promoting tissue

repair, ﬁbrosis, or regeneration [42,44,45].

Macrophages are differentiated from circulating monocytes

which usually arrive at the damaged site 1–3 days after neu-

trophils (Fig. 2A) [46]. Their accumulation will often peak at 4–

7 days after the injury, although elevated accumulations can be

observed up to 21 days [47]. The two main blood monocyte subsets

in the mouse are the Ly6C

CX3CR1

mid

CCR2

(CD62L

CD43

low

) and

the Ly6C

low

CX3CR1

CCR2



(CD62L



CD43

) monocytes [48]

(human equivalents are the CD14

and the CD14

low

CD16

mono-

cytes). There is some evidence to suggest that the primary function

of Ly6C

low

cells is to survey endothelial integrity [49,50]. By con-

trast, Ly6C

monocytes represent ‘‘classical monocytes” that are

recruited to sites of inﬂammation [48].

The two main chemokines/related receptors involved in the

inﬂammation-dependent recruitment of monocyte subsets from

blood, bone marrow and spleen are CCL2/CCR2 and CX3CL1/

CX3CR1 (Fig. 2B) [51,52]. For instance, ﬁbroblast, epithelial, and

endothelial cells surrounding the injured tissue produce CCL2, in

response to DAMPs and inﬂammatory cytokines. Interestingly,

depending on the tissue, one or both monocyte subsets are

recruited. For example, only Ly6C

monocytes are recruited from

the circulation in muscle injury models [53,54]. They ﬁrst acquire

16 Z. Julier et al. / Acta Biomaterialia 53 (2017) 13–28

an inﬂammatory function and further mature into Ly6C

low

macro-

phages with repair functions. However, after myocardial infarction,

both monocyte subsets appear to home in the injured tissue at dif-

ferent stages of inﬂammation via CCR2 and CX3CR1, respectively

[55]. The Ly6C

subset ﬁrst inﬁltrates the infarcted heart and exhi-

bits inﬂammatory functions, while the Ly6C

low

subset is recruited

at a later stage and stimulates repair by expressing high amounts

of VEGF-A and by promoting deposition of collagen.

Driving the recruitment of different monocyte populations,

both CCR2 and CX3CR1 appear to be essential for proper healing

in several tissues. For example, Cx3cr1

/

mice display reduced

levels of

-smooth muscle actin and collagen, reduced neovascu-

larization as well as delayed healing in skin wounds [56]. Similarly,

the loss of CX3CR1 leads to delayed skeletal muscle repair [57].

Moreover, deﬁciency in the CCL2–CCR2 axis appears to impair

muscle and skin repair [58]. For instance, Eming and colleagues

have shown that CCR2 is critical for the recruitment of Ly6C

CCR2

monocytes to skin wounds, leading to proangiogenic macrophages

crucial for vascularization [59]. Interestingly, the study showed

that macrophages are the main source of VEGF-A in early tissue

repair.

Pro-inﬂammatory macrophages – the so-called ‘‘M1” macro-

phages – may become polarized towards a variety of alternatively

activated anti-inﬂammatory ‘‘M2” macrophages [42]. Although

pro-inﬂammatory and anti-inﬂammatory macrophages are the

two most frequently investigated phenotypes in studies of tissue

healing, macrophages exhibiting tissue repair, pro-ﬁbrotic, anti-

ﬁbrotic, pro-resolving, and tissue regeneration characteristics are

also commonly mentioned in the literature [42]. Indeed, the M1

and M2 nomenclature originate from in vitro characterization

where the M1 phenotype is produced by exposure to IFN-

and

TNF-

, while the M2 phenotype is produced by IL-4 or IL-13 [60].

In this review, we adopted the new classiﬁcation system proposed

by Murray et al. [61] where nomenclature is linked to the activa-

tion standards i.e., M(IFN-

), M(IL-4), M(IL-10), and so forth.

Generally, M(IL-4) macrophages are considered as tissue repair

macrophages, since they express several wound healing factors

such as arginase, ECM components and growth factors such as

VEGF-A, platelet-derived growth factor (PDGF) and insulin-like

growth factor (IGF) [42,56,60] (Fig. 2B). Yet, the mechanisms that

drive macrophages to adopt various tissue repair phenotypes

in vivo are still under intense debate [42,60]. Indeed, macrophage

phenotype associated markers may be expressed simultaneously,

making in vivo characterization even more challenging [63].In

addition to cytokines, microRNAs (miRNA), which control messen-

ger RNA translation and degradation (e.g. messenger RNAs of

cytokines and transcription factors), are most likely critical regula-

tors of macrophage polarization [62,63]. More speciﬁcally, miR-9,

miR-127, miR-155, and miR-125b have been shown to promote

M(IFN-

) polarization while miR-21, miR-124, miR-223, miR-34a,

let-7c, miR-132, miR-146a, and miR-125a-5p support M(IL-4)

polarization in macrophages by targeting various transcription fac-

tors and adaptor proteins [62,63].

While inﬂammatory macrophages can exacerbate tissue injury

and impair tissue healing, persistent activation or sustained mobi-

lization of M(IL-4) macrophages has been hypothesized to con-

tribute to the development of pathological ﬁbrosis [42] (Fig. 2C).

For example, the pro-ﬁbrotic function of M(IL-4) macrophages

has been attributed to their production and activation of TGF-b1

in models of pulmonary ﬁbrosis [64]. In addition to producing

pro-ﬁbrotic mediators, M(IL-4) macrophages have been shown to

directly enhance the survival and activation of myoﬁbroblasts,

which are key cells producing ECM in all organs [65]. Pro-ﬁbrotic

M(IL-4) macrophages also produce signiﬁcant amount of matrix

metalloproteinases (MMPs), and some of which serve as essential

drivers of ﬁbrosis [66].

Macrophages may also be anti-inﬂammatory/anti-ﬁbrotic and

they are thought to be critical for the resolution of most tissue

injury inﬂammation responses. IL-10 – an immunoregulatory cyto-

kine produced by a variety of cell types, including T helper 2 cells

(Th2), regulatory T (Treg) cells and macrophages – is known to

function as a critical anti-inﬂammatory mediator [67]. In addition,

anti-inﬂammatory macrophages regulate the development and

maintenance of IL-10- and TGF-b1-producing Tregs, which

contribute to the resolution of inﬂammatory responses in multiple

tissues (Fig. 2D) [68]. Nevertheless, beside the expansion of IL-10-

induced anti-inﬂammatory macrophages, other mechanisms have

also been shown to trigger anti-inﬂammatory macrophages [42].

For example, IL-6 and IL-21 have been found to enhance IL-4R

expression on macrophages and contribute to the development

of anti-inﬂammatory and anti-ﬁbrotic macrophage function fol-

lowing stimulation with IL-4 or IL-13 [69,70].

Interestingly, it has been recently demonstrated that macro-

phages are critical for the regeneration (i.e. the full restoration of

the tissue function) of various tissues [71–73]. For example,

Godwin and colleagues found that macrophages are essential for

limb regeneration in adult salamanders [71]. Moreover, mice can

regenerate cardiac tissue until seven days post-birth and it has

been demonstrated that monocytes and macrophages are required

for the cardiac regeneration process. Remarkably, proﬁling of car-

diac macrophages from regenerating and non-regenerating hearts

indicated that neonatal macrophages have a unique polarization

that does not ﬁt into M(IFN-

) or M(IL-4) phenotypes [73].

Importantly, it remains unclear whether an individual macro-

phage (recruited or tissue-resident) is capable of adopting all the

phenotypes at different time in response to the injured tissue

microenvironment, or if distinct subsets of monocytes and macro-

phages are committed to adopt the various phenotypes [42,60]. For

instance, in several tissues such as the central nervous system and

the liver, macrophages switch from a pro-inﬂammatory phenotype

to a repair phenotype where IL-4, IL-10 and phagocytosis play crit-

ical roles in the conversion [74–76]. In the context of skin injury,

chemokines (e.g. CX3CL1) drives circulating CX3CR1

monocytes

trafﬁc into the damaged site. The CX3CR1

monocytes become M

(IL-4)-like macrophages and secrete factors such as VEGF-A,

TGF-b[56], IL-13, IL-10 and several chemokines [77].

Overall, monocytes and macrophages can exacerbate inﬂamma-

tion, promote tissue repair and ﬁbrosis, or drive regeneration.

While the detailed mechanisms regulating macrophage functions

during tissue healing are still unclear, their critical role in the

repair and regeneration processes marks them as a primary target

when designing regenerative strategies.

2.4. Pericytes

Pericytes are ubiquitous mural cells of blood microvessels,

which facilitate the initial extravasation of immune cells from

the blood [78]. Pericytes are also a source of stem/progenitor cells

and they secrete multiple growth factors and cytokines, as well as

other soluble mediators [79] (Fig. 2D). For example, they con-

tribute to skeletal muscle regeneration by driving immune cells

to cross the endothelium [80] and they are most likely a source

of myogenic precursors [80]. Pericytes also contribute to tissue

healing by promoting angiogenesis at the damaged site [79]. For

instance, injection of pericytes into mouse cardiac tissue after

infarction improves the healing process, by reducing scar forma-

tion, ﬁbrosis and cardiomyocyte apoptosis via secretion of angio-

genic factors and miRNA [81]. These examples demonstrate that

pericytes are pro-regenerative cells, but they are also source of

scar-forming myoﬁbroblasts in several organs, including skin, liver,

and in the central nervous system [1,79]. In addition, pericytes

interact with the immune cells involved in the scarring process.

Z. Julier et al. / Acta Biomaterialia 53 (2017) 13–28 17

For example, they induce the mobilization of Ly6C

monocytes

that further stimulate scar formation by secreting factors such as

TGF-b1, TNF-

, and PDGFs (Fig. 2C). These factors induce pericytes

to change their morphology leading to vascular permeability, pro-

liferation and expression of tissue inhibitors of metalloproteinases

(TIMPs) [82]. Therefore, pericytes have the capacity to support

regeneration, but in acute or chronic inﬂammation their regenera-

tive function can switch to a ﬁbrotic function. Consequently, one

should design strategies to promote the regenerative capacity of

pericytes (i.e. differentiation into functional tissue cells), while

avoiding promotion of their differentiation into myoﬁbroblasts.

2.5. Dendritic cells

In a manner similar to macrophages, dendritic cells (DCs) will

phagocytise particles and process danger signals at the injury site.

Although their precise role during tissue repair and regeneration

remains not fully understood [83], studies show that they play

an important role in the tissue healing process [26,83–85]. For

example, it has been shown that plasmacytoid DCs sense skin

injury via host-derived nucleic acids (recognized by TLR7 and

TLR9) and promote wound healing through type I interferons

[26]. Burn wound closure is also signiﬁcantly delayed in DC-

deﬁcient mice [83]. The impaired wound healing seems to be asso-

ciated with signiﬁcant suppression of early cellular proliferation,

granulation tissue formation, wound levels of TGF-b1 and forma-

tion of blood vessels. In addition, in a myocardial infarction model,

DC-depleted mice show impaired ventricular functions and remod-

eling, with particularly high levels of inﬂammatory cytokines along

with an unbalanced M(IFN-

):M(IL-4) macrophages ratio strongly

tilted towards M(IFN-

)[85]. DCs most likely act as an

immunoregulator during tissue healing through control of macro-

phages homeostasis.

2.6. T cells

Growing evidence points towards T cells playing a crucial role

in tissue repair and regeneration. While interesting mechanisms

have been revealed, the exact function of the different T cell types

and subsets and their level of accumulation at injury sites are lar-

gely unknown and seem to vary from tissue to tissue. The majority

bT cell fraction appears to have both pro- and anti-regenerative

sub-populations. Meanwhile, the minority tissue resident

dT cell

fraction has been widely reported as being pro-regenerative [86–

89].

T cells are capable of secreting a diverse range of cytokines and

growth factors, which have beneﬁcial or inhibitory effects on tissue

healing (Fig. 2C, D). In the context of bone, there is evidence that

both CD4

(T helper 1, Th1) and CD8

(cytotoxic) T cell subsets

inhibit regeneration [90,91]. For example, fracture healing is accel-

erated in Rag1

/

mice (a mouse model without functional T and B

cells) [92] or when CD8

T cells are actively depleted [90].Ona

mechanistic level, it has been demonstrated that T cells inhibits

MSC-driven bone formation in the mouse via IFN-

and TNF-

[91]. Similar research in humans showed that secretion of IFN-

and TNF-

by effector memory CD8

T cells can result in delayed

osteogenesis and fracture healing [90]. On the other hand, studies

have shown that CD4

Tregs are critical for the repair and regener-

ation of several tissues including skin [93], bone [91,94], lungs

[95–97], kidney [98,99], skeletal muscle [100,101], and cardiac

muscle [102]. For example, after damage to mouse skeletal

muscles, Tregs can comprise up to 50% of the T cell population

between day 14 and 30 [100]. The presence of Tregs results in

the production of arginase [103] and anti-inﬂammatory cytokines

such as IL-10 and TGF-b[94]. These secreted factors provide an

anti-inﬂammatory microenvironment conducive to repair and

polarization of macrophages [94]. Even as conventional T cells

move away, Treg levels remain elevated. This may be because

Tregs that reside in visceral adipose, muscle and lamina propria

express epithelial growth factor receptor (EGF-R) [104,105]. The

expression of EGF-R allows the growth factor amphiregulin

secreted by mast cells to maintain Tregs at the damaged site

[104]. Once present, Tregs proliferate and upregulate amphiregulin

secretion, which is necessary for regeneration [100].

The

dT cells are also important in the tissue healing process.

For example, both humans and mice do not heal skin wounds as

fast or effectively in the absence of

dT cells [106]. Functionally,

the pro-repair insulin-like growth factor-1 (IGF-1) is produced by

both mouse [107] and human [108]

dT cells. In the context of tis-

sue healing, the dendritic epithelial

dT cells (DETCs) are the most

well characterized

dsubset [88]. DETCs have an unusual

dendritic-like morphology in the mouse skin, and they respond

within hours to skin tissue damage by secreting chemokines and

TNF-

to attract macrophages [88]. Additionally, DETCs accelerate

tissue repair by secreting growth factors and cytokines such as IGF-

1, KGF-1 (FGF-7), KGF-2 (FGF-10), IL-22, and IL-17A [88]. For

instance, it has been shown that

dT cells peak between 2 and

7 days after bone injury in the mouse and secrete IL-17A, which

enhance osteoblast functions [87]. Additionally,

d-derived IL-22

prompts proliferation and migration of epithelial cells in various

tissues [109]. Overall,

dT cells play both a central role in recruiting

innate immune cells as well as directly stimulating tissue growth.

We have made signiﬁcant headway in understanding the

importance of T cells during tissue repair and regeneration, in

particular Tregs and

dT cells. Treg and

dT cells secreted growth

factors and cytokines are most likely critical for to orchestrate

tissue healing, particularly in skin and muscle. Nevertheless, the

mechanisms by which the different T cell types and their respec-

tive subsets modulate the immune response to tissue injury are

still very elusive. In addition, T cells probably directly interact with

tissue resident stem or progenitor cell populations, and this could

be a useful niche to exploit for designing new regenerative

strategies.

2.7. B-cells

There is little available evidence on the role of B cells in tissue

healing. Given the origin of B cells within the bone marrow, it

would be expected that there would be cross talk between B cells

and bone tissue [110]. For example, IgM

B cells are important in

repair by secreting osteoprotegerin to accelerate bone regeneration

[111]. Interestingly, while CD4

T cells help upregulate osteoprote-

gerin via the CD40/CD40L pathway, CD8

T cells in contrast inhibit

osteoprotegerin expression [111]. As noted above, mice deﬁcient in

both T and B cells have faster bone healing, suggesting depletion of

the adaptive immune system as a promising strategy to augment

bone regeneration. However, we would argue that there is still

much to be discovered regarding the role of B cells in the repair

and regeneration of various tissues.

3. Promoting tissue regeneration by modulating the immune

system

In the ﬁrst part of this review, we have seen that the immune

system greatly inﬂuences tissue repair and regeneration in both

negative and positive fashion. Therefore, controlling the immune

regulations of tissue healing is becoming an attractive avenue in

regenerative medicine, and the design of regenerative strategies

may progress in parallel with our understanding of the crosstalk

between immune components, stem/progenitor cells and the

tissue healing process. In the next sections, we highlight different

18 Z. Julier et al. / Acta Biomaterialia 53 (2017) 13–28

approaches that attempted controlling the immune system to pro-

mote tissue repair or regeneration. In many cases, these

approaches are based on biomaterials or use biomaterials as deliv-

ery systems for immune modulators (Fig. 3).

3.1. Immune modulation by the physicochemical properties of

biomaterials

Implanted biomaterials can have a signiﬁcant intrinsic effect on

the immune system and macrophage polarization, either promot-

ing or reducing inﬂammation depending on their physicochemical

properties. The form that the biomaterial takes (solid, hydrogel or

micro/nanoparticles), the level of crosslinking and the degradabil-

ity, the hydrophobicity, the topography, and the nature of the bio-

material (synthetic vs naturally derived) are important parameters

to consider (Fig. 3)[112,113]. Synthetic biomaterials that have

been used to modulate the immune response following tissue

injury are for example poly lactic-co-glycolic acid (PLGA), poly(lac-

tic acid) (PLA), and polyethylene glycol (PEG). Naturally derived

biomaterials are for instance decellularized tissues such as human

or porcine skin or porcine small intestine submucosa (SIS), or fab-

ricated scaffolds made of natural molecules such as collagen, ﬁbrin,

hyaluronic acid, chitosan, alginate or silk.

Illustrating the importance of the biomaterial crosslinking, scaf-

folds with a high level of crosslinking usually drive a predomi-

nantly inﬂammatory macrophage response [112,113]. For

example, it has been demonstrated that SIS implantation in rat

preferentially induced anti-inﬂammatory macrophages while a

carbodiimide crosslinked form of SIS induces predominantly

inﬂammatory macrophages [114]. Similarly, macrophages seeded

on non-crosslinked porcine dermis or non-crosslinked porcine

SIS, produces lower levels of IL-1, IL-6, and IL-8 compared to

macrophages seeded on chemically cross-linked porcine dermis

[115].

The surface chemistry also appears to inﬂuence macrophage

adhesion and their cytokine secretion proﬁle. For example, neu-

trally charged hydrophilic-modiﬁed polymers have been shown

to promote less macrophage and less foreign body giant cell forma-

tion compared to hydrophobic and ionic surfaces [116]. Although

there were fewer cells on the hydrophilic/neutral surface, the

macrophages were further activated to produce signiﬁcantly

greater amounts of cytokine (IL-1, IL-6, IL-8, and IL-10) than

hydrophobic and ionic surfaces [116].

When designing a biomaterial, modulating the surface topogra-

phy is an interesting method to regulate the cellular response via

control of cell shape and elasticity. The modulation of macrophage

function, phenotype and polarization to varying topography has

been a subject of research for several decades [112]. Studies on

the role of topography on macrophage polarization strongly sug-

gest an advantage of stimulating macrophage elongation for pro-

moting anti-inﬂammatory polarization [117]. This can be

achieved by micro-patterning the surface and to control attach-

ment, or could be achieved by patterning macrophage ligands on

the surface to promote elongation of cells [112].

A number of naturally derived biomaterials such as high

molecular weight hyaluronic acid [118] and chitosan [119], which

have radical oxygen species-scavenging properties, have intrinsic

anti-inﬂammatory properties. Nevertheless, in the case of most

Fig. 3. Strategies based on biomaterials and drug delivery systems to promote tissue regeneration by controlling the immune system. Biomaterial-based strategies aiming at

improving the healing process through immunomodulation can be achieved either by the biomaterial itself and/or by the delivery of immunomodulators. Strategies most

commonly aim either at the delivery of pro-inﬂammatory modulators to initiate the healing process or the delivery of anti-inﬂammatory modulators to promote the

resolution phase via anti-inﬂammatory/anti-ﬁbrotic macrophages. More complex strategies rely on a sequential delivery of pro-inﬂammatory and anti-inﬂammatory

molecules to exert a more comprehensive control over the tissue healing process. Black arrows indicate a differentiation path. Red arrows indicate induction. Blue arrows

indicate inhibition. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

Z. Julier et al. / Acta Biomaterialia 53 (2017) 13–28 19

biomaterials, loading or functionalization of the biomaterial with

anti-inﬂammatory molecules is necessary to modulate the inﬂam-

matory microenvironment. Naturally derived biomaterials such as

collagen and ﬁbrin are ideal for releasing immune modulators

through enzyme-mediated degradation. On the other hand, syn-

thetic materials may allow for increased control over degradation

and release kinetics of therapeutics, with the caveat that the bio-

material itself and its degradation products should cause a mini-

mal response when implanted [115].

3.2. Immune modulation by decellularized ECM

Excised tissues can be processed to separate cells from the ECM,

leaving only a decellularized ECM scaffold. The structure of these

natural scaffolds inﬂuences numerous cellular processes and can

be used to create a pro-regenerative environment [120]. Moreover,

with the ECM proteins being highly conserved across species,

xenografts are usually well tolerated [121], limiting the risk of

undesired inﬂammation which could interfere with the regulation

of the immune environment. Indeed, among other properties,

decellularized ECM has shown to modulate the wound immune

microenvironment through macrophage polarization [114], with

the ability to direct macrophages towards either an M(IFN-

)or

M(IL-4) phenotype. This immune modulation usually depends on

the composition and structure of the scaffold. Although the exact

underlying mechanism is still not fully elucidated [122], a recent

study suggests that the effects could be carried out by matrix-

bound microvesicles (MBVs) embedded in the ECM [123]. The

study by Badylak and colleagues showed that MBVs were biologi-

cally active and were partially responsible for the effect of the scaf-

fold. Indeed, after isolation from urinary bladder matrix, MBVs

were able to stimulate neurite extension on neuroblastoma cells.

A potential mediator of this activity are miRNAs present within

MBVs. Interestingly, although a certain number of miRNA were

conserved across multiple MBVs of different source, a signiﬁcant

amount was tissue-speciﬁc and could partially explain the differ-

ent effects induced by decellularized scaffolds depending on their

tissue of origin.

Interestingly, the ability to control inﬂammation through

macrophage polarization allows xenograft of acellular ECM to be

more beneﬁcial than an autologous transplantation in some cases.

For example, in a model of tendon reconstruction in mice, the use

of decellularized urinary bladder matrix induced a greater migra-

tion of progenitor cells towards reconstructed tendons compared

to autologous grafts [124]. This improved mobilization of progeni-

tor cells seems to be attributed to an anti-inﬂammatory M(IL-4)-

like response induced by the decellularized ECM scaffold. Indeed,

it has been extensively shown that transplantation of acellular

scaffolds usually results in an M(IL-4)-like response with less scar-

ring compared to cellular scaffolds [122]. In addition, it has been

recently demonstrated that tissue-derived ECM scaffolds induce a

pro-regenerative immune environment through a robust Th2

immune response, which drive macrophage polarization towards

an M(IL-4) phenotype via IL-4 [125].

Importantly, the type of response induced by a decellularized

ECM scaffold highly depends on the source tissue were the ECM

was harvested. Indeed, a study comparing the macrophage

response after being exposed to ECM derived from different types

of tissue showed a very heterogenous behavior [126]. In this study,

SIS, urinary bladder matrix, brain ECM, esophageal ECM, and colo-

nic ECM all induced an M(IL-4) response while dermal ECM

induced an M(IFN-

) phenotype. Interestingly, ECMs derived from

liver, and skeletal muscle did not induce a particular macrophage

phenotype.

Decellularized ECMs also present an interesting option for the

delivery of immunomodulatory molecules. For instance, decellu-

larized bones have been used for the sequential release of two

types of cytokines, the pro-inﬂammatory IFN-

and the anti-

inﬂammatory IL-4 [127]. This sequential release promoted macro-

phage transition from a M(IFN-

) to M(IL-4) phenotype and

enhanced vascularization of the bone scaffolds in a murine subcu-

taneous implantation model.

3.3. Delivery of inﬂammatory molecules

There is a large emphasis on enhancing tissue repair by down-

regulating unwanted inﬂammation. However, pro-inﬂammatory

molecules including danger signals and pro-inﬂammatory cytoki-

nes are necessary to start the tissue healing program. For instance,

the delivery of heat shock protein 70, an endogenous agonist of

TLR2 and TLR4 [128], accelerates wound healing by up-regulating

macrophage-mediated phagocytosis [129]. Similarly, activation of

TLR9 using CpG has been shown to promote skin repair in primates

[130]. These examples demonstrate that the principle of using pro-

inﬂammatory molecules to treat tissue damage could work in

some cases (Fig. 3). Indeed, the inﬂammatory chemokine stromal

cell-derived factor-1 (SDF-1, CXCL12) and prostaglandin E2

(PGE2) have been extensively explored in tissue repair and

regeneration.

3.3.1. SDF-1

SDF-1 is an inﬂammatory and pro-angiogenic chemokine that

has been shown to be very important in the tissue healing process

[131], in particular by its capacity to mobilize progenitor cells

[132]. For instance, both human and mouse MSCs express CXCR4,

a SDF-1 receptor, allowing the cells to trafﬁc towards SDF-1

[133,134]. A large number of studies have used biomaterials such

as silk-collagen [135], gelatin [136], alginate [137], PEGylated ﬁb-

rin [138], poly(lactic-co-glycolic acid) [139], and thiol functional-

ized sP(EO-stat-PO) [140] to deliver SDF-1 in a controlled

manner, both to increase angiogenesis and recruit CXCR4

cells,

including macrophages [141], hematopoietic stem cells [132] and

MSCs [139]. Biomaterials delivering SDF-1 have been used for

many tissue types and the usefulness of this strategy has been

demonstrated in tendons [135], cardiac muscle [138,140], skin

[136] and liver models [132]. Nevertheless, one challenge to using

SDF-1 is its sensitivity to protease, as the cytokine is cleaved by

MMP-2 and serine exopeptidase CD26. This unwanted protein

degradation can be overcome by modifying the MMP-2/CD26

cleavage sites or by codelivering enzymes inhibitors such as saxa-

gliptin [132]. A second concern may be that SDF-1 is implicated in

macrophage-driven hypertrophic scar formation. Indeed, cells such

as mouse lung ﬁbrocytes and pro-ﬁbrotic pericytes have also been

shown to trafﬁc towards SDF-1 in vivo [79,97]. Therefore, appropri-

ate SDF-1 dosing is important when designing therapies, to avoid

induction of ﬁbrosis.

3.3.2. PGE2

Prostaglandin E2 (PGE2) is part of a family of pro-inﬂammatory

lipid molecules known as prostanoids [142]. PGE2 and its multiple

receptors (EP1, EP2, EP3 and EP4) have been involved in both pro-

and anti-regenerative functions. For example, elevated levels of

PGE2 are found in periodontal disease [143]. Conversely, PGE2

can increase bone formation [142,144] and angiogenesis [145].

Within the immune system, PGE2 can induce proliferation of T

cells and cause their apoptosis [142]. Interestingly, while being

pro-inﬂammatory, PGE2 has also been shown to inhibit prolifera-

tion and skews the immune response to Th2 [142] by inhibiting

IL-12 [146], IFN-

[147] and IL-2 [148] secretion by human

lymphocytes.

While PGE2 can be beneﬁcial for tissue healing, PGE2 therapy

requires multiple doses and has signiﬁcant side effects, making it

20 Z. Julier et al. / Acta Biomaterialia 53 (2017) 13–28

a poor therapeutic [144,149]. As such limiting PGE2 locally using

biomaterial delivery systems would be better than repeated sys-

temic administration of PGE2. For example, PGE2 in a cholesterol

bearing pullulan nanogel was effective in building bone in mice

[150]. Nanogels could be further improved by replacing PGE2 with

an agonist that only binds one of its four receptors. Two studies

using an BMP-2/EP4 agonist combination in either a PEG nanogel

[149] or polylactic acid gel [151] were successful in inducing bone

repair or mineralization respectively in mice. Thus, using an ago-

nist to a speciﬁc PGE2 receptor such as EP4 in combination with

growth factors slowly released via a biomaterial may be an effec-

tive therapy.

3.4. Delivery of anti-inﬂammatory molecules

Although inﬂammation at the site of tissue injury is necessary

to kick-start the healing response, its resolution is crucial to

advance the healing process and to restore tissue integrity. The

pro-inﬂammatory function of macrophages is essential during

the early stages of inﬂammation, but proper tissue healing requires

macrophages to be polarized towards an anti-inﬂammatory

phenotype. The pro-resolving activity of macrophages notably

includes the development and maintenance of Tregs. Tregs in turn

contribute to creating an anti-inﬂammatory environment

beneﬁcial to tissue repair and help sustain the anti-inﬂammatory

phenotypes of macrophages (Fig. 2D). The mechanisms inducing

the passage from a pro-inﬂammatory state to a resolution state

naturally exist, but therapeutic strategies aiming at promoting this

transition can further improve the healing process (Fig. 3). For

example, polymer particles fabricated from poly (cyclohexane-

1,4-diylacetone dimethylene ketal) were loaded with an inhibitor

of p38, a mitogen-activated protein kinases important for immune

cell activation, to diminish the post-infarction inﬂammatory

response in the myocardium [152]. In a myocardial infarction

model, the particles signiﬁcantly reduced superoxide and TNF-

production, and resulted in a reduction of ﬁbrosis as well as

improved cardiac function.

3.4.1. Pro-resolving mediators

Resolvins, protectins, lipoxins and maresins secreted by phago-

cytes, are specialized pro-resolving mediators derived from omega

3 fatty acids [153,154], limiting both the recruitment of neu-

trophils and their ingestion by macrophages [39]. For example,

pro-resolving mediators upregulate the expression of CCR5 (a

receptor for inﬂammatory chemokines such as CCL3 and CCL5)

by senescent neutrophils and activated T cells. Thus, CCR5

apop-

totic leukocytes sequester inﬂammatory chemokines and act as

terminators of their signaling during the resolution of inﬂamma-

tion [155]. A resolvin-based strategy has already proved to be efﬁ-

cient at promoting wound healing in a model of obese diabetic

mice through enhanced resolution of peritonitis [156]. Similarly,

administration of protectin on wounds in the same diabetic mouse

model also improved reepithelization and the formation of granu-

lation tissue as well as innervation [157]. Injections of resolvin and

lipoxin have also been shown to be able to control the macrophage

polarization induced after a chitosan scaffold implantation [158].

Indeed, although chitosan usually induces inﬂammatory

macrophages when the degree of acetylation exceeds 15% [159],

injections of lipoxin or resolvin were able to shift the polarization

balance towards a anti-inﬂammatory phenotype in a mouse air-

pouch model.

3.4.2. Inhibitors of TNF-

The pro-inﬂammatory activity of M(IFN-

) macrophages is lar-

gely mediated by the release of TNF-

. While this cytokine has

been shown to positively regulate tissue repair and regeneration

in some situations, its excess can impair the healing process. For

example, pathological levels of TNF-

may induce osteoclastogen-

esis (via T cell secretion of RANKL which activates RANK on osteo-

clasts) resulting in more bone reabsorption than osteogenesis.

Thus, strategies aiming at blocking the activity of TNF-

have been

proposed to diminish the effect of the pro-inﬂammatory macro-

phages. Local delivery of common painkillers including aspirin

[160], ibuprofen [161] and pentoxifylline [162] have shown

encouraging results in reducing TNF-

. For example, simply deliv-

ering aspirin locally with hydroxyapatite/tricalcium phosphate

ceramic particles could reduce TNF-

and prevent apoptosis of

transplanted MSCs, resulting in more bone regeneration [91].

Other strategies include directly targeting TNF-

with TNF-

anti-

bodies. For example, a delivery system based on chitosan/collagen

scaffold has been developed [163], in which a glucose-sensitive

delivery system was capable of releasing TNF-

antibodies upon

increase of glucose level in a diabetic rat model, a condition often

associated with alveolar bone destruction and high level of TNF-

The system successfully reduced inﬂammation and promoted alve-

olar bone healing. Other studies have used hyaluronic acid as a

delivery vehicle for anti-TNF-

. Hyaluronic acid can bind CD44

on macrophages and thus provide the anti-TNF signal directly to

the cell producing the cytokine. For example, hyaluronic acid plus

a monoclonal antibody for TNF-

was effective at inducing early

healing in the rats after a burn [164].

Although many studies have focused on inhibition of TNF-

as a

therapy to overcome unwanted inﬂammation and accelerate tissue

healing, it should be noted that TNF-

might be a useful cytokine to

help begin the healing process in some tissues. For example, in a

rat model, pre-stimulation of MSCs with TNF-

increased their

engraftment to myocardial infarct [165]. Additionally, TNF-

enables mobilization of human and mouse MSCs into damaged tis-

sues [166,167]. After bone fracture in the mouse, TNF-

levels peak

at 24 h post-injury, and help recruit pro-regenerative cells such as

MSCs [167]. A second wave of TNF-

expression peaks at about

four weeks after injury and is necessary for endochondrial bone

formation [167]. In other tissues, TNF-

could also play a positive

role as it helps stimulate production of BMP-2 in the context of car-

diac [165] and skin [168] repair.

3.4.3. Inhibitors of the NF-

B pathway

Many DAMPs and inﬂammatory cytokines such as IL-1 and TNF-

induce the NF-

B pathway and there is a growing body of evi-

dence that inhibiting NF-kB may be a viable option to accelerate

the healing of some tissues. For example, mice deﬁcient in IL-1

receptor antagonist (IL-1Ra) show delayed wound healing due to

higher neutrophil recruitment and subsequent NF-

B activation

in ﬁbroblasts resulting in negative regulation of the pro-repair

TGF-bpathway [169]. In addition, targeting NF-

B may aid bone

regeneration. For instance, inhibiting NF-

B in mouse osteoblasts

can increase bone density in an induced osteoporosis model

[170]. Moreover, it was shown that inﬂammatory cytokines such

as TNF-

and IL-17 reduce osteogenesis of mouse MSCs. These

cytokines impair the Wnt/b-Catenin signaling in MSCs, which is

critical for osteogenesis [171]. Co-delivering MSCs on apatite-

coated PLGA scaffold with a small inhibitor of IKKb(which is a

subunit in the kinase enzyme complex part of the upstream

NF-

B signaling) resulted in much more bone formation in vivo

compared to MSCs delivered without inhibitor. Similarly, it was

shown that IL-1bsignaling through the IL1-R1/MyD88 pathway

inhibits mouse MSC proliferation, migration and differentiation

towards osteoblasts [23]. Indeed, MSC response to growth factor

and Wnt signaling were impaired by IL-1b, due to AKT dephospho-

rylation and b-catenin degradation. Mouse calvarial defect treated

with IL1Ra or a MyD88 inhibitor designed to be covalently incorpo-

rated into ﬁbrin hydrogels and to translocate into cells following

Z. Julier et al. / Acta Biomaterialia 53 (2017) 13–28 21

hydrogel remodeling by proteases signiﬁcantly improved bone

regeneration driven by MSCs [23]. Taken together, these studies

indicate that NF-kB inhibition could have a dual positive effect of

reducing inﬂammation while increasing regeneration driven by

MSCs.

3.4.4. Anti-inﬂammatory cytokines

Anti-inﬂammatory cytokines such as IL-4 and IL-10 are critical

for proper tissue repair and regeneration, since they are involved

in M(IFN-

) to M(IL-4) macrophage switching [42]. In particular,

mouse models have convincingly shown that IL-10 is necessary

for scar-free healing [172]. Indeed, fetal mice are able to repair

cutaneous damage scar free via IL-10 dependent mechanisms,

and this ability can be replicated in adult mice that overexpress

IL-10 [172]. The same effect seems to happen in the heart. It has

been shown that ﬁbrosis after infarct in the mouse is considerably

reduced, when IL-10 is delivered through heparin-based coacer-

vate [173]. For regenerative medicine purposes, IL-10 has been

mostly delivered using plasmid DNA and virus vectors [176], but

IL-4 is often delivered as a protein to induce M(IL-4) macrophage

polarization. For example, slow release of IL-4 conjugated to a bone

scaffold via biotin-streptavidin can enhance M(IL-4) macrophage

polarization [127]. In a rat model of in vivo peripheral nerve dam-

age, IL-4 delivered via injectable agarose hydrogel was effective in

increasing the number of M(IL-4) macrophages [174]. Interest-

ingly, IL-4 delivery via this method resulted in much more axons

being regrown after three weeks compared to the controls, sug-

gesting controlled release of IL-4 may help with peripheral nerve

repair via M(IL-4) macrophages. In a different context, it was

shown that IL-4 delivery could potentially reduce bone degrada-

tion after joint replacement [175]. The study showed that poly-

ethylene particles could degrade mouse calvarias in vitro, but this

process was ameliorated via addition of IL-4 [175]. This study sug-

gests that incorporating IL-4 or similar anti-inﬂammatory cytokine

to implanted materials may prevent unwanted side effects of

implants. Overall, the delivery of IL-4 may enhance tissue regener-

ation in various situations via M(IL-4) macrophage induction.

What is remaining to be explored is the direct effect that IL-4 could

have on T cells.

Another interesting anti-inﬂammatory cytokine is TGF-b1,

which is necessary for tissue repair at the earliest stages [176],

although the molecule has inﬂammatory or anti-inﬂammatory

effects depending on the cell type it signals. For example, TGF-b1

can suppress lymphocyte proliferation as well as activity and can

help to induce immune-suppressive Tregs [177]. Nevertheless,

the cytokine is also highly involved in scar formation [176]. How-

ever, TGF-bhas three isoforms (TGF-b1, 2 and 3) and there is evi-

dence showing that TGF-b3 can be harnessed to accelerate

regeneration and avoid scarring [176]. Indeed, TGF-b3 simply

injected alone on incisional wounds in human patients was able

to slightly, but noticeably reduce post-operative scarring [178].

The design of an optimal delivery system for TGF-b3 may therefore

improve its anti-ﬁbrotic capacity in humans.

3.4.5. siRNA

Small interfering RNA (siRNA)-mediated gene silencing offers

an alternative therapeutic strategy to antibodies and chemical-

based inhibitors. A number of studies have demonstrated the

potential of RNA interference to suppress pro-inﬂammatory path-

ways and inﬂammatory cytokines [176,179–182]. The major chal-

lenges for therapeutic use of siRNAs are to develop methods for

delivering siRNAs to the desired cell types in vivo and to escape

from the endosomal compartment [182].

PLGA particles have been used to deliver TNF-

siRNA for treat-

ing inﬂammation associated with rheumatoid arthritis. In a mouse

model of rheumatoid arthritis, the particles resulted in a reduction

of TNF-

production and inﬂammation in the joint. Similarly, PLGA

particles have been used to deliver polyetherimide(PEI)-conjugated

RIII-targeting siRNA to reduce inﬂammation [179]. The system

proved to be efﬁcient in a rat model of temporomandibular joint

inﬂammation with reduction of IL-1 and IL-6. In a remarkable study,

a lipid nanoparticle was used to deliver a therapeutic siRNA that

reduced the accumulation of CCR2 pro-inﬂammatory monocytes

to inﬂamed tissue [180]. The siRNA targeting CCR2 was adminis-

tered systemically, and shown to reduce the infarct size in a myocar-

dial infarction model, reduce inﬂammatory cells in atherosclerotic

lesion, improve the survival of pancreatic islet allografts, and reduce

tumor volume. Similarly, nanoparticle-based RNA interference that

effectively silences ﬁve key adhesion molecules for arterial leuko-

cyte recruitment has been used to prevent complications after acute

myocardial infarction [181]. Simultaneously encapsulating siRNA

targeting intercellular cell adhesion molecules 1 and 2, vascular cell

adhesion molecule 1, and E- and P-selectins into polymeric

endothelial-avid nanoparticles reduced the recruitment of neu-

trophil and monocyte after myocardial infarction into atheroscle-

rotic lesions and decreased matrix degrading plaque protease

activity. The ﬁve-gene combination RNA interference also curtailed

leukocyte recruitment to ischemic myocardium. Overall, these

studies emphasize the potential of siRNA as a therapeutic to control

the immune system and to reduce the detrimental effect of exces-

sive inﬂammation during the tissue healing process.

3.4.6. miRNA

miRNAs play an important role in immunity [183–185] and tis-

sue healing [186–188]. Their ability to regulate the immune sys-

tem on multiple levels is of particular interest here as they

appear to be involved in the development and functions of

hematopoietic stem cells, as well as innate and adaptive immune

cells. For example, miRNAs can direct macrophages polarization

through targeting of the IRF/STAT pathway, promoting inﬂamma-

tion and its resolution [62]. Moreover, miRNAs can induce Tregs

[189] and regulate many other aspects of the T cell response

[190] via modulation of TCR signaling [191] and T helper cells plas-

ticity [192]. For instance, miR-181a has the ability to initially help

activate mature T cells through increased TCR signaling sensitivity,

but also to later repress this activation through downregulation of

CD69, a promoter of T cell proliferation [196].

Although therapeutic strategies based on the delivery of miR-

NAs are still scarce, studies have shown that their use can be ben-

eﬁcial to tissue healing [193]. For example, in a rat skeletal muscle

injury model, the combined injection of three different miRNAs

improved muscle regeneration while preventing ﬁbrosis [194].

Nevertheless, direct injections of miRNA present limitations such

as in vivo stability or biodistribution, which could be overcome

by the development of advanced delivery systems [193,195].As

for siRNA, biomaterial-based delivery systems are necessary to

optimize the delivery of miRNA [196]. For instance, hydrogels have

been successfully used for ex vivo delivery of miRNAs to cells in 3D

culture [197] and could provide an alternative to soluble injections

for in vivo delivery at a speciﬁc site. Nanoparticle are also a good

option for in vivo delivery of miRNA. For example, delivery of

miR-146a using PEI nanoparticles was able to inhibit renal ﬁbrosis

through suppression of the inﬁltration of F4/80

macrophages

[198]. Currently, options are also being pursued to modulate miR-

NAs signaling in vivo, either by overexpression or inhibition. How-

ever, improvements in delivery methods of the modulators are also

required [193].

3.5. Extracellular vesicles

Extracellular vesicles (EVs), which includes exosomes (from the

endosomal compartment), microvesicles (formed by budding of

22 Z. Julier et al. / Acta Biomaterialia 53 (2017) 13–28

the plasma membrane), and apoptotic bodies (from dying cells),

are phosphor lipid vesicles from 30 nm to 1

m in diameter, used

as cargo container by cells to exchange biomolecules such as trans-

membrane receptors and genetic information [199]. More speciﬁ-

cally, their payload can include cytokines, morphogens, MMPs,

antigens, DNA, non-coding RNAs, mRNAs, and miRNAs, with the

latter being particularly explored in term of the potential func-

tional roles of exosomes [199]. EVs are released by most cell types

and have been detected in all bodily ﬂuids. Once released in the

extracellular milieu, EVs are taken up by the target cells within

the local microenvironment or carried to distant sites though bio-

logical ﬂuids [200].

EVs are most likely important regulators of immune cell

activity and could therefore modulate tissue repair and regener-

ation via the immune system (Fig. 3)[201,202]. EVs derived

from immune cells have been principally studied in the context

of the immune response itself, in particular for cancer

immunotherapy [203]. In the context of tissue healing, there

are few studies looking into the potential of immune cell-

derived EVs. Nevertheless, EVs from immune cells most likely

have a role in the crosstalk between immunity and tissue haling

[201,202]. On the other hand, the most studied EVs in tissue

repair and regeneration have been MSC-derived EVs, whose

functions include immunomodulation [204]. For example, MSC-

derived EVs have been explored as a treatment for ﬁbrotic liver

disease [205]. It was demonstrated that the delivery of exosomes

into the liver reduced ﬁbrosis through inhibition of the TGF-b1

signaling pathway, as well as through inhibition of epithelial-

to-mesenchymal transition of hepatocytes. This effect was most

likely mediated by miR-125b [206]. MSC-derived exosomes can

also inhibit macrophage activation by suppressing TLR signaling

[207]. In a mouse model of hypoxic pulmonary hypertension, it

was shown that the intravenous delivery of MSC-derived exo-

somes suppresses hypoxic inﬂammation by inhibition of pro-

proliferative pathways via miR-17 [208].

In most of the animal in vivo studies, EVs have been delivered by

intravenous or intraperitoneal injection every few days [201,202].

Following injection, studies reported very variable half-life for

vesicles clearance, ranging from 10 min to 12 h [199]. However,

there is currently no evidence that EV administration following tis-

sue injury preferentially homes to the damaged sites [201,202].To

overcome this issue, researchers can use delivery systems to con-

trol the release of EVs in situ. For example, a commercially available

hydrogel (HyStem-HP) has been used to deliver MSC-derived EVs

in a rat critical size bone defect model [209]. In addition, EVs can

be regarded as natural drug delivery vehicles and be loaded with

exogenous therapeutic agents. Since EVs are effective natural sys-

tems for polynucleotide (siRNA, miRNA) and protein delivery,

one can engineer EVs surface to deliver speciﬁc content to speciﬁc

cell types. For instance, siRNA have been delivered by engineered

EVs to suppress pro-inﬂammatory genes [210]. Targeting was

achieved by engineering dendritic cell-derived exosomes to dis-

play Lamp2b, an exosomal membrane protein, fused to the

neuron-speciﬁc RVG peptide and loaded with exogenous siRNA

by electroporation. The therapeutic potential of exosome-

mediated siRNA delivery was demonstrated in mice, by the signif-

icant mRNA and protein knockdown of a therapeutic target in Alz-

heimer’s disease (BACE1). Similar approaches could be used to

engineer EVs, in order to promote tissue regeneration via immune

modulation.

Overall, EVs are certainly a potential therapeutic for promoting

tissue healing via immune modulation. Furthermore, while a

recent study has demonstrated that EVs are able to stay in decellu-

larized tissue scaffolds [126], new researches should explore novel

methods to deliver EVs and to integrate them into other biomate-

rial scaffolds.

3.6. Codelivery of immune modulators and morphogens

Because the tissue healing process involves numerous immune

and morphogenetic signals operating at the same time or sequen-

tially, the delivery of multiple immune and morphogenetic factors

in a spatiotemporal-controlled manner is most likely required to

induce an effective regenerative microenvironment. When deliver-

ing multiple factors, the ﬁrst difﬁculty is to understand which fac-

tors should be delivered at which concentration and at what time.

Then, the challenge is to develop systems able to deliver the differ-

ent factors in a spatiotemporally controlled manner.

One interesting approach is to stimulate an M(IFN-

) macro-

phage response, which resolves rapidly to transition to a M(IL-4)

response [112]. In that regard, Alvarez et al. reviewed multiple

strategies to sequentially release different molecules that polarize

the response to M(IFN-

), then re-polarize to M(IL-4), resulting in

improved healing [211]. For example, decellularized bones were

used as a scaffold and further engineered to sequentially release

IFN-

for inducing a M(IFN-

) response and IL-4 to transit to a M

(IL-4) response [127]. The authors were able to conﬁrm the

sequential stimulation of both macrophage phenotypes and

observed increased vascularization in functionalized scaffold com-

pared to empty scaffolds in a murine subcutaneous implantation

model.

Codelivering growth factors and immune modulators is also a

promising approach. For instance, platelet rich plasma (PRP),

which contain high level of growth factors, has been delivered in

l-lactic acid grafted gelatin with macrophage attracting micelles

containing sphingosine-1-phosphate agonist (SEW2871) to

increase bone regeneration in mice [212]. Interestingly,

SEW2871-micelles and PRP enhanced the level of TNF-

3 days

after application, and increased the anti-inﬂammatory cytokines

IL-10 at day 10. Using the same system, SDF-1 codelivered with

SEW2871-micelles was able to more than double the rate of mouse

skin wound closure. Most likely, the combination of SDF-1/

SEW2871 was able to recruit both MSCs and macrophages to the

damaged skin [134]. A similar approach opted for dual delivery

of FGF-2 and IL-10 via poly(ethylene argininylaspartate diglyceride

(PEAD)/heparin coacervate biomaterial in a mouse myocardial

infarct model [173]. The study showed that the combination of

FGF-2 and IL-10 is more effective than delivering the growth factor

and the cytokine alone. Heart function was restored and ﬁbrosis

was signiﬁcantly reduced. In the context of bone, codelivery of

BMP-2 and EP4 agonist in either a PEG nanogel [149] or polylactic

acid gel [151] was successful in inducing bone regeneration.

4. Conclusion and future perspectives

A remarkable number of immune cells and immune modulators

participate in all phases of the tissue healing process. Therefore

controlling the immune system, in particular key immune cell sub-

sets, is a very plausible strategy to promote tissue regeneration.

However, the complex mechanisms by which the immune system

orchestrates various organs and tissues are still vastly unknown.

For instance, while neutrophils are the ﬁrst circulating immune

cells mobilized after tissue injury, their role in the healing process

has been somewhat overlooked. They are likely involved in macro-

phage polarization, although we still do not know exactly in what

way. Controlling neutrophil mobilization and functions could be an

interesting strategy to promote tissue regeneration.

Macrophages have shown to be critical during most phases of

the tissue healing process, but the mechanisms by which they

change phenotypes to stimulate tissue repair, ﬁbrosis or full regen-

eration remain unclear. Thus, further effort is required to under-

stand the contributions of the different macrophage populations

Z. Julier et al. / Acta Biomaterialia 53 (2017) 13–28 23

and activation states in multiple organ systems. For instance,

inﬂammatory macrophages mature into anti-inﬂammatory macro-

phages in certain type of tissues, while a distinct population of

anti-inﬂammatory macrophages is mobilized in others. Therefore,

depending on the tissue or organ targeted, one could develop

regenerative strategies aimed at stimulating macrophage polariza-

tion or aiming at recruiting pro-wound healing macrophage sub-

sets. The current approaches points towards driving macrophages

polarization to M(IL-4)-like phenotypes, using a variety of immune

modulators delivered through biomaterials and drug delivery

systems. However, one should remember that, although large

numbers of M(IFN-

) macrophages exacerbate tissue injury, per-

sistent activation or sustained recruitment of particular M(IL-4)

macrophage subsets contribute to the development of pathological

ﬁbrosis. One of the keys could be to reveal the exact mechanisms

that drive the expansion of anti-inﬂammatory/anti-ﬁbrotic macro-

phages in vivo and further implement these mechanisms into

regenerative strategies. Perhaps, the answer will come from the

situation in neonates, which display macrophage populations with

pro-regenerative capacities distinct from the adult. For instance,

altering key macrophage transcription factors that stabilize or

induce these particular pro-regenerative populations could hold

promise to stimulate regeneration.

As with macrophage subsets, there is growing evidence that T

cell subsets can have both anti-regenerative and pro-regenerative

properties. Yet, the mechanisms driving T cell mobilization, activa-

tion and conversion at sites of tissue injury are still largely

unknown. We have seen that Tregs are immune-suppressive and

particular Treg populations are pro-regenerative. Th2 and

dT cells

could also be critical for inducing a pro-regenerative environment.

Therefore, biomaterials and the delivery of immunomodulators

could be exploited to modulate T cell activities and promote regen-

eration. One could induce T cell conversion into Tregs from con-

ventional T cells recruited at a site of injury or promote the

mobilization of natural Tregs using chemokines. For example,

CCR4, the receptor for CCL22 chemokine, is typically expressed

more on Tregs compared to conventional T cells [213,214]. One

study was able to successfully reduce inﬂammation and periodon-

tal disease in mice and dogs via injection of CCL22 microparticle

powder [214]. Other options could include increasing the amount

of anti-inﬂammatory cytokines typically produced by Tregs, Th2

cells and M(IL-4) macrophages such as IL-4, IL-10 or IL-13.

The aging of the immune system is also a parameter that could

be considered when designing regenerative therapies. Indeed, the

baseline macrophage polarization states and the activity of T cell

subsets may be affected by patient characteristics [42,215]. Along

with differences in the number of stem/progenitor cells in neo-

nates and adults, there is now increasing evidence for changes in

macrophage phenotypes and T cell activities with age and diseases.

Therefore, alterations in the crosstalk between tissue-resident or

transplanted stem/progenitor cells and macrophages and/or T cells

could have major impacts on tissue regeneration after injury and in

aging.

Today, ample evidence suggests that an active control of the

immune system is a very plausible therapeutic strategy to induce

tissue regeneration. However, because we still have sparse knowl-

edge about the immune mechanisms modulating the tissue healing

process, one of the main challenges is to target the right immune

cell populations and pathways for the particular tissue or organ

that needs to be regenerated. Then, the challenge is to engineer

efﬁcient biomaterial and delivery system platforms for controlling

the immune-mediated mechanisms of tissue healing. The next

generation of regenerative strategies may evolve from typical

biomaterial-, stem cell-, or growth factor-centric approaches to

an immune-centric approach, seeking to control the immune sys-

tem as a means of promoting regrowth of tissues and organs.

Acknowledgements

This work was supported in part by the research grant of Astel-

las Foundation for Research on Metabolic Disorders to M.M.M.

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Proteomic analysis and functional validation reveal distinct therapeutic capabilities related to priming of mesenchymal stromal/stem cells with IFN-γ and hypoxia: potential implications for their clinical use

Article

Full-text available

May 2024

Mesenchymal stromal/stem cells (MSCs) are a heterogeneous population of multipotent cells that can be obtained from various tissues, such as dental pulp, adipose tissue, bone marrow and placenta. MSCs have gained importance in the field of regenerative medicine because of their promising role in cell therapy and their regulatory abilities in tissue repair and regeneration. However, a better characterization of these cells and their products is necessary to further potentiate their clinical application. In this study, we used unbiased high-resolution mass spectrometry-based proteomic analysis to investigate the impact of distinct priming strategies, such as hypoxia and IFN-γ treatment, on the composition and therapeutic functionality of the secretome produced by MSCs derived from the amniotic membrane of the human placenta (hAMSCs). Our investigation revealed that both types of priming improved the therapeutic efficacy of hAMSCs, and these improvements were related to the secretion of functional factors present in the conditioned medium (CM) and exosomes (EXOs), which play crucial roles in mediating the paracrine effects of MSCs. In particular, hypoxia was able to induce a pro-angiogenic, innate immune response-activating, and tissue-regenerative hAMSC phenotype, as highlighted by the elevated production of regulatory factors such as VEGFA, PDGFRB, ANGPTL4, ENG, GRO-γ, IL8, and GRO-α. IFN-γ priming, instead, led to an immunosuppressive profile in hAMSCs, as indicated by increased levels of TGFB1, ANXA1, THBS1, HOMER2, GRN, TOLLIP and MCP-1. Functional assays validated the increased angiogenic properties of hypoxic hAMSCs and the enhanced immunosuppressive activity of IFN-γ-treated hAMSCs. This study extends beyond the direct priming effects on hAMSCs, demonstrating that hypoxia and IFN-γ can influence the functional characteristics of hAMSC-derived secretomes, which, in turn, orchestrate the production of functional factors by peripheral blood cells. This research provides valuable insights into the optimization of MSC-based therapies by systematically assessing and comparing the priming type-specific functional features of hAMSCs. These findings highlight new strategies for enhancing the therapeutic efficacy of MSCs, particularly in the context of multifactorial diseases, paving the way for the use of hAMSC-derived products in clinical practice.

Loss of the ability to regenerate body appendages in vertebrates: from side effects of evolutionary innovations to gene loss

Article

May 2024
BIOL REV

The ability to regenerate large body appendages is an ancestral trait of vertebrates, which varies across different animal groups. While anamniotes (fish and amphibians) commonly possess this ability, it is notably restricted in amniotes (reptiles, birds, and mammals). In this review, we explore the factors contributing to the loss of regenerative capabilities in amniotes. First, we analyse the potential negative impacts on appendage regeneration caused by four evolutionary innovations: advanced immunity, skin keratinization, whole-body endothermy, and increased body size. These innovations emerged as amniotes transitioned to terrestrial habitats and were correlated with a decline in regeneration capability. Second, we examine the role played by the loss of regeneration-related enhancers and genes initiated by these innovations in the fixation of an inability to regenerate body appendages at the genomic level. We propose that following the cessation of regenerative capacity, the loss of highly specific regeneration enhancers could represent an evolutionarily neutral event. Consequently, the loss of such enhancers might promptly follow the suppression of regeneration as a side effect of evolutionary innovations. By contrast, the loss of regeneration-related genes, due to their pleiotropic functions, would only take place if such loss was accompanied by additional evolutionary innovations that compensated for the loss of pleiotropic functions unrelated to regeneration, which would remain even after participation of these genes in regeneration was lost. Through a review of the literature, we provide evidence that, in many cases, the loss in amniotes of genes associated with body appendage regeneration in anamniotes was significantly delayed relative to the time when regenerative capability was lost. We hypothesise that this delay may be attributed to the necessity for evolutionary restructuring of developmental mechanisms to create conditions where the loss of these genes was a beneficial innovation for the organism. Experimental investigation of the downregulation of genes involved in the regeneration of body appendages in anamniotes but absent in amniotes offers a promising avenue to uncover evolutionary innovations that emerged from the loss of these genes. We propose that the vast majority of regeneration-related genes lost in amniotes (about 150 in humans) may be involved in regulating the early stages of limb and tail regeneration in anamniotes. Disruption of this stage, rather than the late stage, may not interfere with the mechanisms of limb and tail bud development during embryogenesis, as these mechanisms share similarities with those operating in the late stage of regeneration. Consequently, the most promising approach to restoring regeneration in humans may involve creating analogs of embryonic limb buds using stem cell-based tissue-engineering methods, followed by their transfer to the amputation stump. Due to the loss of many genes required specifically during the early stage of regeneration, this approach may be more effective than attempting to induce both early and late stages of regeneration directly in the stump itself.

Clinical Applications of Adipose-Derived Stem Cell (ADSC) Exosomes in Tissue Regeneration

Article

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May 2024
INT J MOL SCI

Adipose-derived stem cells (ADSCs) are mesenchymal stem cells with a great potential for self-renewal and differentiation. Exosomes derived from ADSCs (ADSC-exos) can imitate their functions, carrying cargoes of bioactive molecules that may affect specific cellular targets and signaling processes. Recent evidence has shown that ADSC-exos can mediate tissue regeneration through the regulation of the inflammatory response, enhancement of cell proliferation, and induction of angiogenesis. At the same time, they may promote wound healing as well as the remodeling of the extracellular matrix. In combination with scaffolds, they present the future of cell-free therapies and promising adjuncts to reconstructive surgery with diverse tissue-specific functions and minimal adverse effects. In this review, we address the main characteristics and functional properties of ADSC-exos in tissue regeneration and explore their most recent clinical application in wound healing, musculoskeletal regeneration, dermatology, and plastic surgery as well as in tissue engineering.

Macrophages modulate fibrosis during newt lens regeneration

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Full-text available

May 2024

Background Previous studies have suggested that macrophages are present during lens regeneration in newts, but their role in the process is yet to be elucidated. Methods Here we generated a transgenic reporter line using the newt, Pleurodeles waltl, that traces macrophages during lens regeneration. Furthermore, we assessed early changes in gene expression during lens regeneration using two newt species, Notophthalmus viridescens and Pleurodeles waltl. Finally, we used clodronate liposomes to deplete macrophages during lens regeneration in both species and tested the effect of a subsequent secondary injury after macrophage recovery. Results Macrophage depletion abrogated lens regeneration, induced the formation of scar-like tissue, led to inflammation, decreased iris pigment epithelial cell (iPEC) proliferation, and increased rates of apoptosis in the eye. Some of these phenotypes persisted throughout the last observation period of 100 days and could be attenuated by exogenous FGF2 administration. A distinct transcript profile encoding acute inflammatory effectors was established for the dorsal iris. Reinjury of the newt eye alleviated the effects of macrophage depletion, including the resolution of scar-like tissue, and re-initiated the regeneration process. Conclusions Together, our findings highlight the importance of macrophages for facilitating a pro-regenerative environment in the newt eye by regulating fibrotic responses, modulating the overall inflammatory landscape, and maintaining the proper balance of early proliferation and late apoptosis of the iPECs.

Engineering extracellular vesicles for ROS scavenging and tissue regeneration

Article

Full-text available

Jun 2024

Stem cell therapy holds promise for tissue regeneration, yet significant challenges persist. Emerging as a safer and potentially more effective alternative, extracellular vesicles (EVs) derived from stem cells exhibit remarkable abilities to activate critical signaling cascades, thereby facilitating tissue repair. EVs, nano-scale membrane vesicles, mediate intercellular communication by encapsulating a diverse cargo of proteins, lipids, and nucleic acids. Their therapeutic potential lies in delivering cargos, activating signaling pathways, and efficiently mitigating oxidative stress—an essential aspect of overcoming limitations in stem cell-based tissue repair. This review focuses on engineering and applying EVs in tissue regeneration, emphasizing their role in regulating reactive oxygen species (ROS) pathways. Additionally, we explore strategies to enhance EV therapeutic activity, including functionalization and incorporation of antioxidant defense proteins. Understanding these molecular mechanisms is crucial for optimizing EV-based regenerative therapies. Insights into EV and ROS signaling modulation pave the way for targeted and efficient regenerative therapies harnessing the potential of EVs.

Bioactive Patch for Rotator Cuff Repairing via Enhancing Tendon‐to‐Bone Healing: A Large Animal Study and Short‐Term Outcome of a Clinical Trial

Article

Full-text available

Jun 2024

Tissue engineering has demonstrated its efficacy in promoting tissue regeneration, and extensive research has explored its application in rotator cuff (RC) tears. However, there remains a paucity of research translating from bench to clinic. A key challenge in RC repair is the healing of tendon–bone interface (TBI), for which bioactive materials suitable for interface repair are still lacking. The umbilical cord (UC), which serves as a vital repository of bioactive components in nature, is emerging as an important source of tissue engineering materials. A minimally manipulated approach is used to fabricate UC scaffolds that retain a wealth of bioactive components and cytokines. The scaffold demonstrates the ability to modulate the TBI healing microenvironment by facilitating cell proliferation, migration, suppressing inflammation, and inducing chondrogenic differentiation. This foundation sets the stage for in vivo validation and clinical translation. Following implantation of UC scaffolds in the canine model, comprehensive assessments, including MRI and histological analysis confirm their efficacy in inducing TBI reconstruction. Encouraging short‐term clinical results further suggest the ability of UC scaffolds to effectively enhance RC repair. This investigation explores the mechanisms underlying the promotion of TBI repair by UC scaffolds, providing key insights for clinical application and translational research.

From Time to Timer in Wound Healing Through the Regeneration

Chapter

Jun 2024
ADV EXP MED BIOL

Hard-to-heal wounds are an important public health issue worldwide, with a significant impact on the quality of life of patients. It is estimated that approximately 1–2% of the global population suffers from difficult wounds, which can be caused by a variety of factors such as trauma, infections, chronic diseases like diabetes or obesity, or poor health conditions. Hard-to-heal wounds are often characterized by a slow and complicated healing process, which can lead to serious complications such as infections, pressure ulcers, scar tissue formation, and even amputations. These complications can have a significant impact on the mobility, autonomy, and quality of life of patients, leading to an increase in healthcare and social costs associated with wound care. The preparation of the wound bed is a key concept in the management of hard-to-heal wounds, with the aim of promoting an optimal environment for healing. The TIME (Tissue, Infection/Inflammation, Moisture, Edge) model is a systematic approach used to assess and manage wounds in a targeted and personalized way. The concept of TIMER, expanding the TIME model, further focuses on regenerative processes, paying particular attention to promoting tissue regeneration and wound healing in a more effective and comprehensive way. The new element introduced in the TIMER model is “Regeneration”, which highlights the importance of activating and supporting tissue regeneration processes to promote complete and lasting wound healing. Regenerative therapies can include a wide range of approaches, including cellular therapies, growth factors, bioactive biomaterials, stem cell therapies, and growth factor therapies. These therapies aim to promote the formation of new healthy tissues, reduce inflammation, improve vascularization, and stimulate cellular proliferation to accelerate wound closure and prevent complications. Thanks to continuous progress in research and development of regenerative therapies, more and more patients suffering from difficult wounds can benefit from innovative and promising solutions to promote faster and more effective healing, improve quality of life, and reduce the risk of long-term complications.

Physical Properties and Biochemical Composition of Extracellular Matrix-Derived Hydrogels Dictate Vascularization Potential in an Organ-Dependent Fashion

Article

May 2024
ACS APPL MATER INTER

Towards Stem Cell Therapy for Critical-Sized Segmental Bone Defects: Current Trends and Challenges on the Path to Clinical Translation

Article

Full-text available

May 2024

The management and reconstruction of critical-sized segmental bone defects remain a major clinical challenge for orthopaedic clinicians and surgeons. In particular, regenerative medicine approaches that involve incorporating stem cells within tissue engineering scaffolds have great promise for fracture management. This narrative review focuses on the primary components of bone tissue engineering—stem cells, scaffolds, the microenvironment, and vascularisation—addressing current advances and translational and regulatory challenges in the current landscape of stem cell therapy for critical-sized bone defects. To comprehensively explore this research area and offer insights for future treatment options in orthopaedic surgery, we have examined the latest developments and advancements in bone tissue engineering, focusing on those of clinical relevance in recent years. Finally, we present a forward-looking perspective on using stem cells in bone tissue engineering for critical-sized segmental bone defects.

Navigating the Immunological Crossroads: Mesenchymal Stem/Stromal Cells as Architects of Inflammatory Harmony in Tissue-Engineered Constructs

Article

Full-text available

May 2024

In the dynamic landscape of tissue engineering, the integration of tissue-engineered constructs (TECs) faces a dual challenge—initiating beneficial inflammation for regeneration while avoiding the perils of prolonged immune activation. As TECs encounter the immediate reaction of the immune system upon implantation, the unique immunomodulatory properties of mesenchymal stem/stromal cells (MSCs) emerge as key navigators. Harnessing the paracrine effects of MSCs, researchers aim to craft a localized microenvironment that not only enhances TEC integration but also holds therapeutic promise for inflammatory-driven pathologies. This review unravels the latest advancements, applications, obstacles, and future prospects surrounding the strategic alliance between MSCs and TECs, shedding light on the immunological symphony that guides the course of regenerative medicine.

MSCs-Derived Exosomes: Cell-Secreted Nanovesicles with Regenerative Potential

Article

Full-text available

Aug 2016

Exosomes are membrane-enclosed nanovesicles (30–150 nm) that shuttle active cargoes between different cells. These tiny extracellular vesicles have been recently isolated from mesenchymal stem cells (MSCs) conditioned medium, a population of multipotent cells identified in several adult tissues. MSCs paracrine activity has been already shown to be the key mediator of their elicited regenerative effects. On the other hand, the individual contribution of MSCs-derived exosomes for these effects is only now being unraveled. The administration of MSCs-derived exosomes has been demonstrated to restore tissue function in multiple diseases/injury models and to induce beneficial in vitro effects, mainly mediated by exosomal-enclosed miRNAs. Additionally, the source and the culture conditions of MSCs have been shown to influence the regenerative responses induced by exosomes. Therefore, these studies reveal that MSCs-derived exosomes hold a great potential for cell-free therapies that are safer and easier to manipulate than cell-based products. Nevertheless, this is an emerging research field and hence, further studies are required to understand the full dimension of this complex intercellular communication system and how it can be optimized to take full advantage of its therapeutic effects. In this mini-review, we summarize the most significant new advances in the regenerative properties of MSCs-derived exosomes and discuss the molecular mechanisms underlying these effects.

Matrix-bound nanovesicles within ECM bioscaffolds

Article

Full-text available

Jun 2016

Biologic scaffold materials composed of extracellular matrix (ECM) have been used in a variety of surgical and tissue engineering/regenerative medicine applications and are associated with favorable constructive remodeling properties including angiogenesis, stem cell recruitment, and modulation of macrophage phenotype toward an anti-inflammatory effector cell type. However, the mechanisms by which these events are mediated are largely unknown. Matrix-bound nanovesicles (MBVs) are identified as an integral and functional component of ECM bioscaffolds. Extracellular vesicles (EVs) are potent vehicles of intercellular communication due to their ability to transfer RNA, proteins, enzymes, and lipids, thereby affecting physiologic and pathologic processes. Formerly identified exclusively in biologic fluids, the presence of EVs within the ECM of connective tissue has not been reported. In both laboratory-produced and commercially available biologic scaffolds, MBVs can be separated from the matrix only after enzymatic digestion of the ECM scaffold material, a temporal sequence similar to the functional activity attributed to implanted bioscaffolds during and following their degradation when used in clinical applications. The present study shows that MBVs contain microRNA capable of exerting phenotypical and functional effects on macrophage activation and neuroblastoma cell differentiation. The identification of MBVs embedded within the ECM of biologic scaffolds provides mechanistic insights not only into the inductive properties of ECM bioscaffolds but also into the regulation of tissue homeostasis.

TOLL-LIKE RECEPTOR 4 PLAYS AN ESSENTIAL ROLE IN EARLY SKIN WOUND HEALING (180.1)

Article

May 2012

Toll-like receptor 4 (TLR4) plays a key role in the initiation of innate immunity and in the regulation of adaptive immune responses. Using microarray analysis and PCR, TLR4 expression was observed to increase in experimental murine skin wounds at the early stages, suggesting a role in wound inflammation. Closure of excisional wounds was significantly delayed in TLR4 deficient (C3H/HeJ) as compared to wild type mice, and both IL-1β and IL-6 production were significantly lower in the wounds of TLR4 deficient mice. EGF also markedly decreased in the wound edge of epidermis in TLR4 deficient mice. In vitro studies confirmed that a wound stimulus induces TLR4 mRNA expression in primary normal human epidermal keratinocytes (NHEK). In vitro injury also induced the phosphorylation of p38 and JNK MAPK, as well as the expression of IL-1β and TNF-α by NHEK. Blockade of TLR4 abolished the phosphorylation of p38 and JNK MAPK, and blockade of TLR4 and/or p38/JNK abolished IL-1β production. The results strongly suggest that inflammatory cytokine production by injured NHEK is stimulated via the TLR4-p38 and JNK MAPK signaling pathway. Together, the current results provide evidence for a role of TLR4 at sites of injury, and suggest that TLR4 is an important regulator of wound inflammation.

Research on rapeseed plant modeling and optimization from 3D point cloud

Conference Paper

Jul 2015

As one of the most important oil crops, the high-accuracy rapeseed digital model contributes to agronomic horticulture, crop-breeding and plant genetics research. How to build a three-dimensional (3D) plant model, especially a high-quality leaf model, has great significance to the research on the rapeseed plant growth status. In this paper, we present a three-dimensional oilseed rape plant reconstruction method based on point cloud data. Firstly, we provide a novel approach to reconstruct 3D rapeseed shape model by utilizing the acquired 3D point clouds, and the original point cloud acquired via 3D laser scanning should be preprocessed, the main idea of processing includes de-noising and simplifying the original point clouds. Then the meshing method and mesh optimization process have been carried out to attain a high-quality 3D rapeseed plant mesh model. Thus the rapeseed plants can be non-destructively monitored by measuring the reconstructed rapeseed plant models, and we can obtain the needed parameters such as the leaf area. Experiments are implemented on real datasets, and the results indicate that the accuracy of the leaf area values are highly closed to the real values, which are measured by conventional digital processing methodology.

Extracellular Vesicles: Immunomodulatory messengers in the context of tissue repair/regeneration

Article

Sep 2016
EUR J PHARM SCI

Inflammation is a complex and highly regulated biological process, crucial for a variety of functions in the human body, from host response against infectious agents to initiation of repair/regeneration of injured tissues. In the context of tissue repair, the action of different immune cell populations and their interplay with tissue specific cells, including stem cells, is still being uncovered. Extracellular Vesicles (EV) are small membrane vesicles secreted by cells in a controlled manner, which can act local and systemically. The ability of EV to influence tissue repair and regeneration has been proposed as a physiologically intelligent and targeted strategy of cell communication. Herein, the role of EV in tissue repair is reviewed, summarising first their contribution to the regulation of immune cell function, and discussing the implications for the resolution of inflammation during repair. Next, the impact of EV on cell proliferation and differentiation, and on extracellular matrix remodelling, key aspects of the subsequent phases of tissue repair, is addressed. Finally, EV-based therapies are discussed, focusing on the application of naturally produced EV, and the use of EV as delivery vehicles.

Solubilized Extracellular Matrix Bioscaffolds Derived from Diverse Source Tissues Differentially Influence Macrophage Phenotype

Article

Sep 2016
J BIOMED MATER RES A

The host response to biomaterials is a critical determinant of their success or failure in tissue-repair applications. Macrophages are among the first responders in the host response to biomaterials and have been shown to be predictors of downstream tissue remodeling events. Biomaterials composed of mammalian extracellular matrix (ECM) in particular have been shown to promote distinctive and constructive remodeling outcomes when compared to their synthetic counterparts, a property that has been largely attributed to their ability to modulate the host macrophage response. ECM bioscaffolds are prepared by decellularizing source tissues such as dermis and small intestinal submucosa. The differential ability of such scaffolds to influence macrophage behavior has not been determined. The present study determines the effects of ECM bioscaffolds derived from eight different source tissues upon macrophage surface marker expression, protein content, phagocytic capability, metabolism, and antimicrobial activity. The results show that macrophages exposed to small intestinal submucosa (SIS), urinary bladder matrix (UBM), brain ECM (bECM), esophageal ECM (eECM), and colonic ECM (coECM) express a predominant M2-like macrophage phenotype, which is pro-remodeling and anti-inflammatory (iNOS-/Fizz1+/CD206+). In contrast, macrophage exposure to dermal ECM resulted in a predominant M1-like, pro-inflammatory phenotype (iNOS+/Fizz1-/CD206-), whereas liver ECM (LECM) and skeletal muscle ECM (mECM) did not significantly change the expression of these markers. All solubilized ECM bioscaffold treatments resulted in an increased macrophage antimicrobial activity, but no differences were evident in macrophage phagocytic capabilities, and macrophage metabolism was decreased following exposure to UBM, bECM, mECM, coECM, and dECM. The present work could have important implications when considering the macrophage response following ECM implantation for site-appropriate tissue remodeling.

Circulating extracellular vesicles: Their role in tissue repair and regeneration

Article

Jul 2016
TRANSFUS APHER SCI

Extracellular vesicles (EVs) have been a growing interest of the scientific community in recent years due to the wide possibilities of their evaluation as biomarkers of disease, and their potential to be used as therapeutic agents or vehicles. EVs that circulate in plasma carry proteins and nucleic acids, potentially to distant locations in the body where they can interfere with several cellular processes. To aid understanding of this rapidly evolving field, circulating EVs, including immune cell-derived ones, are reviewed here. Their cellular origins and described functions are discussed in a perspective of their contribution to regenerative processes. Different techniques for EV engineering and examples of their application are reviewed as a strong future direction of EV research. A summary of important aspects yet to be addressed ties up this review.

RNAi targeting multiple cell adhesion molecules reduces immune cell recruitment and vascular inflammation after myocardial infarction

Article

Jun 2016

Myocardial infarction (MI) leads to a systemic surge of vascular inflammation in mice and humans, resulting in secondary ischemic complications and high mortality. We show that, in ApoE(-/-) mice with coronary ligation, increased sympathetic tone up-regulates not only hematopoietic leukocyte production but also plaque endothelial expression of adhesion molecules. To counteract the resulting arterial leukocyte recruitment, we developed nanoparticle-based RNA interference (RNAi) that effectively silences five key adhesion molecules. Simultaneously encapsulating small interfering RNA (siRNA)-targeting intercellular cell adhesion molecules 1 and 2 (Icam1 and Icam2), vascular cell adhesion molecule 1 (Vcam1), and E- and P-selectins (Sele and Selp) into polymeric endothelial-avid nanoparticles reduced post-MI neutrophil and monocyte recruitment into atherosclerotic lesions and decreased matrix-degrading plaque protease activity. Five-gene combination RNAi also curtailed leukocyte recruitment to ischemic myocardium. Therefore, targeted multigene silencing may prevent complications after acute MI.

MicroRNAs as regulatory elements in immune system logic

Article

May 2016
NAT REV IMMUNOL

MicroRNAs as regulatory elements in immune system logic

Article

Apr 2016
NAT REV IMMUNOL

MicroRNAs (miRNAs) are crucial post-transcriptional regulators of haematopoietic cell fate decisions. They act by negatively regulating the expression of key immune development genes, thus contributing important logic elements to the regulatory circuitry. Deletion studies have made it increasingly apparent that they confer robustness to immune cell development, especially under conditions of environmental stress such as infectious challenge and ageing. Aberrant expression of certain miRNAs can lead to pathological consequences, such as autoimmunity and haematological cancers. In this Review, we discuss the mechanisms by which several miRNAs influence immune development and buffer normal haematopoietic output, first at the level of haematopoietic stem cells, then in innate and adaptive immune cells. We then discuss the pathological consequences of dysregulation of these miRNAs.

Promoting Tissue Regeneration by Modulating the Immune System

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