Immunomodulation for Tissue Repair and Regeneration

Abstract

Various immune cells participate in repair and regeneration following tissue injury or damage, orchestrating tissue inflammation and regeneration processes. A deeper understanding of the immune system’s involvement in tissue repair and regeneration is critical for the development of successful reparatory and regenerative strategies. Here we review recent technologies that facilitate cell-based and biomaterial-based modulation of the immune systems for tissue repair and regeneration. First, we summarize the roles of various types of immune cells in tissue repair. Second, we review the principle, examples, and limitations of regulatory T (Treg) cell-based therapy, a representative cell-based immunotherapy. Finally, we discuss biomaterial-based immunotherapy strategies that aim to modulate immune cells using various biomaterials for tissue repair and regeneration.

Modulating the immune system for tissue repair and regeneration

Immune cells for tissue repair and regeneration

Repair of injured tissue requires the successful transition from the inflammation phase to the regeneration phase. In the tissue inflammation phase, invaded pathogens and tissue debris at the wound site are removed by immune cells, which is required for the subsequent tissue regeneration phase. The tissue regeneration phase induces progenitor cells to rebuild injured wound sites in an anti-inflammatory environment.

This tissue repair process can be divided into four steps: hemostasis, inflammation, repair and remodeling [1] (Fig. 1). In the hemostasis step, platelets accumulate at the wound site and blood clotting is initiated with fibrin. Platelets and wound debris generate damage-associated molecular patterns (DAMPs), pathogen-associated molecular patterns (PAMPs), and various chemokines and these signals recruit immune cells to the wound site, starting the inflammation step. In the inflammation step, inflammatory immune cells remove tissue debris and pathogens while stimulating progenitor cells, myofibroblasts and epithelial cells. The inflammation step then ceases and the repair and remodeling steps begin. These processes take place over a longer period of time than prior steps, and involve the inhibition of tissue inflammation by anti-inflammatory or reparative immune cells and the induction of progenitor cells, proliferation of stem cell proliferation, and accumulation of collagen for new tissue generation [2].

Fig. 1.

Steps of the tissue repair process and immune cells in chronological order. Reprinted with permission from ref [1].

In short, hemostasis and inflammation steps contribute to tissue inflammation while repair and remodeling steps induce tissue regeneration and stabilize newly generated tissue. Late conversion to the tissue regeneration phase may result in chronic inflammation and tissue damage, while a prolonged regeneration phase may lead to pathogenic fibrosis, scarring, or organ failure [3]. Therefore, an appropriate conversion between tissue repair steps is necessary. Various immune cells, including neutrophils, Natural Killer (NK) cells, macrophages, T cells, and B cells, participate in these tissue repair steps, modulating the tissue microenvironment. These immune cells sequentially arrive at injury sites and activate or suppress one another, resulting in orchestrated tissue repair. Therefore, a profound understanding of immune cells in tissue repair is needed. Here, immune cells in the tissue repair process and their crucial roles are listed and explained below.

Macrophages

Macrophages are known to play a central role in tissue regeneration, and their phenotypes and roles have been extensively investigated and revealed. Macrophages are largely divided into M1 and M2 phenotypes according to their inflammatory activities. First, monocytes infiltrate into wound sites along with neutrophils and NK cells, mostly by the CCR2 chemokine-dependent pathway [4]. When monocytes arrive at the injured site, the tissue injury site is predominated by inflammatory signals such as DAMPs, PAMPs, and proinflammatory cytokines by other immune cells, including interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). This proinflammatory environment induces infiltrating monocytes to differentiate into proinflammatory M1 macrophages [5, 6]. These M1 macrophages engulf pathogens and tissue debris and produce proinflammatory cytokines, including IFN-γ, interleukin (IL)-1β and IL-6. In addition to phagocytosis and cytokine release, M1 macrophages participate in adaptive immunity as antigen presenting cells (APCs). High expression of major histocompatibility complex (MHC) IIand costimulatory molecules including CD80 and CD86 enables antigen presentation by macrophages. Therefore, M1 macrophages further induce inflammation against pathogens in tissue injury sites. M1 macrophages present processed antigens and produce proinflammatory cytokines geared towards the generation of T helper 1 (Th1) cells, thereby enhancing the inflammatory phase at tissue injury [7].

A prolonged inflammatory state at injury sites may be destructive to tissue and so the regeneration phase should be promptly initiated at an appropriate time point. M1 macrophages are converted to anti-inflammatory M2 macrophages by various signals such as IL-4, IL-10, and IL-13 [8]. These M2 macrophages activate anti-inflammatory immune cells such as T helper 2 (Th2) cells and Treg (Treg) cells, and secrete anti-inflammatory cytokines such as IL-10 and IL-4 and growth factors including transforming growth factor-beta (TGF-β), transforming growth factor-alpha (TGF-α), and platelet-derived growth factor (PDGF). [9] These cytokines in turn promote angiogenesis, progenitor cell proliferation, and extracellular matrix (ECM) remodeling [10, 11] for tissue regeneration.

Neutrophils

Neutrophils are the first immune cells to arrive at the injury site and play a critical role in the inflammatory phase. These cells are recruited by DAMPs, PAMPs, and cytokines released from tissue-residing cells [12, 13]. Neutrophils boost tissue inflammation by releasing proinflammatory cytokines such as IFN-γ, IL-1β and IL-6, induce monocyte infiltration, and induce M1 polarization [14]. In addition, neutrophils kill and phagocytose pathogens by reactive oxygen species (ROS), proteases, antimicrobial peptides, and neutrophil extracellular traps (NETs) [13]. Therefore, neutrophils are central immune modulating cells in the inflammatory phase of tissue repair.

On the other hand, prolonged neutrophil activation may lead to excessive inflammation in tissue. Therefore, timely neutrophil apoptosis is necessary to mitigate tissue damage [15]. After the inflammation process clears pathogens and tissue debris from the injury site, neutrophil apoptosis is initiated and apoptotic neutrophils are phagocytosed by macrophages. Efferocytosis induces macrophage transition to an anti-inflammatory phenotype, supporting the transition from the proinflammatory phase to the tissue regeneration phase. Neutrophil apoptosis is thus essential for the completion of the inflammatory phase in the tissue repair process. In addition, recent research has also shown the important role of neutrophils in terminating the inflammatory phase. Yang and colleagues showed that neutrophils induced macrophage polarization from proinflammatory M1 macrophages to anti-inflammatory M2 macrophages by ROS production in a mouse liver repair model [16]. Depletion of neutrophils in this liver injury model resulted in impaired M2 transition, and neutrophil transfer of wild-type mice restored M2 transition. This research emphasizes that neutrophils participate in the thorough tissue repair process. In addition, there is evidence that neutrophils participate in the tissue regeneration phase, as neutrophils secrete matrix metalloproteinase (MMP)-9 to initiate angiogenesis [17, 18]. Since neutrophils have both inflammatory and reparative characteristics, it’s been proposed that neutrophils may also be divided into N1/N2 phenotypes, similar to that of M1/M2 macrophages [19]. N1 neutrophils induce inflammation in tissues as previously described and are polarized by lipopolysaccharide (LPS) in vitro. N2 neutrophils, on the other hand, hinder inflammation in tissue sites and induce anti-inflammatory immune cells. As the tissue repair process continues, the number of N1 neutrophils substantially decreases and the number of N2 neutrophils increases in the reparative phase.

NK cells

NK cells are cytotoxic immune cells adept at killing pathogens and possess several similarities with cytotoxic T cells. NK cells arrive at the tissue injury site earlier than cytotoxic T cells, however, in a similar time frame as that of monocytes. NK cells remove pathogen-infected cells by perforin and granzyme B-mediated pathways and secrete IFN-γ, granulocyte–macrophage colony-stimulating factor (GM-CSF), and TNF-α to induce M1 macrophages in tissue injury sites. Moreover, NK cells interact with dendritic cells (DCs) by cytokines, including IFN-γ and TNF-α, and DCs in turn activate NK cells [20]. This mutual activation further enhances inflammation in the tissue injury site. Antigen presentation by activated DCs results in the activation of Th1 cells and these Th1 cells are further stimulated by NK cells secreting IFN-γ.

While NK cells contribute to tissue inflammation, there are some conflicting views on the overall influence of NK cells in the tissue repair process. Some researchers revealed that depletion of NK cells in a tissue injury model resulted in successful tissue repair [21], while overactivation of NK cells resulted in excessive IFN-γ in the liver and therefore impaired the liver repair process [22]. On the other hand, liver-resident hepatic NK cells showed beneficial effects on liver regeneration. Hepatic NK cells induced moderate inflammation by TNF-α and IL-22 secretion and induced proliferation of hepatocytes [23]. Therefore, a better understanding of NK cells in tissue repair is needed before NK cells can be modulated for better tissue repair processes.

Th1 cells and Th2 cells

T cells consist of lymphocytes that can be largely divided into CD4⁺ T cells and CD8⁺ T cells. Among T-cell subsets, cytotoxic CD8⁺ T cells are known to adversely affect tissue regeneration, exacerbating tissue inflammation [24, 25]. On the other hand, CD4⁺ T cells are prominently engaged in tissue repair, as their regenerative effect is revealed in myocardial infarction (MI) [26] and pancreatitis [27]. Among CD4 T cells, Th1 cells, Th2 cells, and Treg cells are known to be associated with the tissue repair process. Th1 cells secrete IFN-γ, TNF-α, and IL-12, enhancing inflammation of tissues to prepare tissue regeneration. Th2 cells secrete IL-4, IL-13, and TGF-β, and these cytokines promote the development of an anti-inflammatory environment. Th2 cells induce M1 to M2 polarization and induce the development of Treg cells. Thus, Th2 cells play essential roles in the tissue regeneration step. However, overactivation of these lymphocytes may result in pathogenic fibrosis [28]. Anti-inflammatory cytokines, especially IL-13, are regulators of fibrosis in tissue recovery. In contrast, Th1 cells secrete MMPs and IFN-γ, inhibiting collagen accumulation in tissue injury sites. Excessive fibroblast activation and subsequent collagen in tissue injury sites result in fibrosis, and pathogenic fibrosis often leads to organ failure. Therefore, a balance between Th1 cells and Th2 cells is required for successful tissue recovery. In addition to Th1 and Th2 cells, recent research revealed that T helper 17 (Th17) cells also take part in the tissue repair process [29–31]. Th17 cells produce IL-17, IL-21, and IL-22. These cytokines induce immune cell recruitment, antimicrobial peptide production, and wound healing, contributing to both tissue inflammation and sequential regeneration.

Treg cells

The Treg cell is a well-known anti-inflammatory immune cell characterized by CD4, CD25, and forkhead Box P3 (Foxp3) expression. Treg cells can suppress tissue inflammation through several mechanisms. Treg cell secretes TGF-β, IL-10, IL-4, and IL-13. These cytokines induce monocyte differentiation toward M2 macrophages, inhibiting overall tissue inflammation [32, 33]. In addition, Treg cells are capable of inhibiting CD4⁺ Th1 cells and CD8⁺ cytotoxic T cells, thereby inhibiting IFN-γ secretion and tissue inflammation [34]. Furthermore, Treg cells can inhibit neutrophil migration to wound sites to signal the end of the inflammatory phase [35]. Surface expression of cytotoxic T lymphocyte antigen-4 (CTLA-4) also contributes to Treg cell-mediated anti-inflammatory activity, as CTLA-4 suppresses costimulatory molecules on APCs in tissue injury sites [36]. Lastly, Treg cells also facilitate tissue repair by inducing the proliferation of progenitor cells and stem cells [37, 38].

Treg cells suppress excessive inflammatory responses and promote tissue regeneration through various pathways. They can also modulate the immune response by interacting with other immune cells (Fig. 2). Treg cells interact with other T cells through both direct and indirect mechanisms [39, 40]. Human Treg cells express high levels of CTLA-4 and produce IL-10, TGF-β, and IFN-γ. They interact with naïve and memory CD4⁺ T cells to inhibit their proliferation [41]. Treg cells suppress pathogenic activation of Th17 cells by IL-10 production [42]. They also interact with cytotoxic CD8⁺ T cells to suppress their inflammatory functions and disturb their proliferation [43]. Additionally, CTLA-4-expressing CD25⁺CD4⁺Treg cells suppress normal T cells even in the absence of CD28 molecules [44]. Treg cells can also interact with various antigen presenting cells, and affect monocytes and macrophages to control their activation and phenotype. Treg cells suppress the immune response of monocytes via Fas/Fas ligand interactions that regulates their survival [45]. Treg cells produce IL-13 which promotes macrophage IL-10 secretion and autocrine/paracrine signaling of macrophages by IL-10 in turn boosts macrophage efferocytosis and reduces inflammation [46]. Treg cells also induce inflammatory M1 macrophage polarization to anti-inflammatory M2 macrophages and achieve resolution of inflammation and regeneration of damaged tissue. Macrophages transferred with CD4⁺CD25⁺Treg cells have upregulated M2 markers such as CD23 and downregulated M1 markers such as CD80 and MHC-II [47]. Human macrophages co-cultured with Treg cells show increase in phagocytic activity and blunted responses to LPS, resulting in a decrease in proinflammatory cytokine production [48]. M2 macrophages can also induce Treg cells by different pathways, which can lead to more effective immune suppression [49]. It is well-known that tolerogenic DCs can induce Treg cells, but Treg cells can also downregulate pro-inflammatory DC activity. Treg cells strongly adhere to DCs which prevents adhesion to other APCs and immune cells [50]. Antigen-specific Treg cells have powerful interactions with DCs, remove complexes of MHC-II and peptides from the surface of DCs in turn, and diminish antigen presenting ability [51]. Treg cells can induce neutrophils to secrete anti-inflammatory cytokines such as IL-10 and TGF-β and suppress IL-6 secretion. They can also decrease neutrophil immune activation with the expression of heme oxygenase-1 (HO-1) and suppressor of cytokine signaling 3 molecule(SOCS3) [52]. Treg cells can affect NK cells like other immune cells using cytokines such as TGF-β or by consuming IL-2 [53, 54]. NK cells suppressed by Treg cells have lower NKG2D, CD107a expression, and IFN-γ secretion [55].

Fig. 2.

Interaction of Treg cells with various immune cells. Reprinted with permission from ref [56].

B cells

The B cells is a subset of lymphocytes that can differentiate into plasma cells to produce antibodies and present antigens to T cells. While little is known about how B cells participate in tissue regeneration, B cells may manipulate tissue immune cells by secreting cytokines and presenting antigens to T cells. In one study, B-cell treatment in wound injury showed fibroblast proliferation, TGF-β increase, and MMP-2 decrease [57]. These changes resulted in faster wound healing in mice, showing the tissue regeneration effect of B cells in wound healing models. On the other hand, activated B cells can secrete inflammatory cytokines TNF-α, IL-1β, and IL-6, which promoted tissue fibrosis in a murine MI model [58]. Therefore, whether B cells contribute to tissue repair is not clearly defined. Further study of B-cell activity in tissue repair would enable modulation of B cells for successful tissue regeneration.

Cell-based immunotherapy

Treg cell-based therapy

Treg cells can modulate immune responses in various parts of the body. They suppress immoderate immune reactions in many tissues and organs. Treg cells can modulate different immune responses in muscle, [59, 60] skin tissues, [61] adipose tissue, [62] intestine, [63] and the central nervous system [64]. Similarly, Treg cells play important roles in the resolution of inflammation, tissue repair, tissue regeneration and many other physiological mechanisms. Therefore, there have been various attempts to use Treg cells to treat different diseases.

Treg cell treatment is generally used in various autoimmune diseases. For example, in a type 1 diabetes mouse model, injection of Treg cells reduced insulitis and resulted in islet-infiltrating lymphocyte apoptosis, thereby alleviating diabetes symptoms [65]. Additionally, DNA methylation of FOXP3 using 5-Aza-2′-deoxycytydine (DAC) increased the number of Treg cells in the thymus and resolved diabetes [66]. In a mouse experimental autoimmune encephalomyelitis (EAE) model, adoptive transfer of self-antigen specific Treg cells reduced central nervous system damage and boosted recovery from the disease [67]. Injection of CTLA-4/dNP2 conjugated chimeric peptide can induce Treg cells and ameliorate disease severity and prevent recurrence [68]. Furthermore, Treg cells are widely used in the treatment of transplant rejection such as graft-versus-host reaction or disease (GvHD) [69]. In a mouse allogenic bone marrow/stem cell transplantation model, in vitro expanded donor Treg cells were transferred to recipient mice and alleviated symptoms [70]. Additionally, a low dose of IL-2 administration was found to regulate Treg cell expansion and resolve disease [71].

Treg cells are also widely used in many inflammatory diseases. For example, research has shown that injection of Treg cells post MI showed therapeutic efficacy through high expression of the anti-inflammatory surface molecule CD73 [72]. Additionally, administration of an antagonist anti-CD28 monoclonal antibody to mice can lead to heart macrophages switching from the M1 phenotype to the M2 phenotype, inducing heart tissue regeneration and reducing the occurrence heart failure [32]. In addition, adoptive transfer of Treg cells in an acute lung injury model can treat the disease via modulation of cytokines and control of neutrophils [73]. Injection of curcumin into acute lung injury model mice induced Treg cells to produce IL-10, leading to macrophage regulation and alleviation of the disease [74]. Rheumatoid arthritis (RA) can also be treated using Treg cell therapies. Collagen-induced arthritis (CIA) mice injected with Treg cells recorded lower arthritis scores and produced low levels of proinflammatory cytokines [75]. Using CTLA-4/IgG fusion protein, the number of Treg cells increased in the joint and spleen, signifying suppression of arthritis symptoms [76]. In addition, various immune cell-related diseases, such as systemic lupus erythematosus [77] and inflammatory bowel disease [78], have been studied with Treg cell treatment.

Similarly, there are two major strategies regarding Treg cell therapies (Fig. 3). One is cell therapy, which involves expanding the number of isolated or induced Treg cells ex vivo, then introducing Treg cells into the body and elevating the anti-inflammatory response to disease treatment. The other method involves drug therapy that induces Treg cells in vivo using various types of drugs. Both cell and drug therapies have their advantages and disadvantages.

Fig. 3.

Various strategies of Treg cell-based therapies. Reprinted with permission from ref [97].

Cell therapies are advantageous in that they can use definitively-induced Treg cells. Ex vivo induced Treg cells can be confirmed by cell analysis before administration. Therefore, many studies using cell therapies have been attempted [70, 73, 75]. However, there are important key factors in cell therapy, particularly when it pertains to Treg cell’s antigen specificity. The antigen specificity of T cells, which is obtained by a complex of (TCR) and a specific antigen, is very important because of the side effects. If T cells are activated in unwanted sites of the body, they will cause an inappropriate immune response and thus can result in incorrect outcomes. Treg cells are much more difficult to proliferate than effector T cells. Thus, to achieve sufficient numbers of Treg cells ex vivo in cell therapy, the polyclonal Treg cell method is frequently used [79]. However, these polyclonal Treg cells do not have antigen specificity, so they can cause side effects. Overall, it is important to obtain an abundant number of Treg cells that have antigen specificity.

Recently, there have been several solutions that use gene-editing and gene delivery technologies. Due to gene editing and delivery systems, researchers can regulate cell protein expression in the fundamental phase. One solution is the delivery of DNA encoding TCR, which recognizes specific antigens [80]. These edited Treg cells are not polyclonal cells but antigen-specific and can also sufficiently proliferate ex vivo. Therefore, several studies have used these gene-edited regulator T cells to treat diseases. In the arthritis model, only TCR receptor editing genes or both TCR receptor and FOXP3 editing genes were transferred to CD4 + CD25 + T cells. They sufficiently expressed TCR and FOXP3, maintaining Treg cell’s properties and relieving arthritis symptoms [81]. Additionally, in a mouse EAE model, Treg cells transduced with myelin basic protein (MBP) peptide were able to treat the disease through suppression of effector T cells [82]. In the T1D model, TCR and FOXP3 edited Treg cells could effectively target islets and alleviate symptoms of the disease [83].

More recently, chimeric antigen receptor (CAR) technology has taken a great part in T cell therapies. CAR, a protein based on a single-chain variable fragment (scFv) in monoclonal antibodies, can recognize specific proteins on target cells with high affinity [84]. Therefore, CAR gene editing technology has also been introduced to Treg cell therapy. Regulatory CAR T therapy is more potent than other antigen-TCR complex therapies because of its high affinity to target antigens and the diversity of antigen types [80]. Therefore, using CAR-encoded DNA, researchers can effectively make regulatory CAR T cells for various diseases [85]. In a mouse EAE model, Treg cells edited with myelin oligodendrocyte glycoprotein(MOG) encoded genes could modulate cytokine release, leading to the relief of EAE symptoms [86]. There were also many studies have treated GvHD with regulatory CAR T cells [87, 88].

In addition, there are various drug therapies for Treg cell based treatment. Various drugs or molecules can be used to induce the Treg cell’s phenotype in vivo. Drugs that can change APC’s properties to immune suppressive phenotypes are widely used to induce Treg cells. For example, tolerogenic DCs induced by curcumin injection can induce Treg cells to inhibit colitis [89]. Treatment with hepatocyte growth factor (HGF) can lead immune cells to tolerogenic DCs and Treg cells [90]. Additionally, other various drugs, such as peptides and small molecules like vitamin D can be used to induce immune-modulating APCs [91, 92]. Proteins such as antibodies and cytokines can also influence the T cell immune response. Oral delivery of anti-CD3 antibody can induce Treg cells and reduce atherosclerosis symptoms [93]. In addition, IL-2/TGF-β treated Treg cells have resistance to changing their phenotype to the inflammatory type [94]. Finally, there are small molecules that alter T cell lifespan and immune profile, such as mammalian target of rapamycin(mTOR) [95, 96].

Neutrophil-based immunotherapy

Neutrophils can remove pathogens in the early immune response. However, prolonged activation and survival of neutrophils have adverse effects on tissue repair and regeneration due to release of excessive inflammatory cytokines. A number of studies have attempted to modulate neutrophil function to achieve tissue repair and regeneration [98]. Neutrophil depletion in myocardial infarction mice exacerbated the myocardial infarction due to lack of neutrophil secretome and neutrophil gelatinase-associated lipocalin [99]. Depletion of neutrophils by injection of Ly6G antibodies to mice with acid-induced acute lung injury hindered alveolar epithelial regeneration [100]. Recently, a study demonstrated that excessive neutrophil extracellular traps (NETs) impaired tissue repair and wound healing by destroying wound structure, disturbing angiogenesis, and reducing regenerative cell functions [101]. Therefore, control of NETs would be a promising strategy for tissue repair and regeneration. Another study has demonstrated that wound healing was promoted when NET functions were suppressed [102]. Also, injection of bastroxobin, a NET inhibitor, improved tissue repair in a mouse model of limb ischemia [103]. These studies indicate that suppression of neutrophil function can promote tissue regeneration and wound healing.

Macrophage-based immunotherapy

Following tissue injury, excessive and prolonged activation of proinflammatory M1 macrophages impairs tissue repair and regeneration, while pro-regenerative M2 macrophages promote tissue repair and regeneration. Thus, timely switching of macrophage phenotype from M1 to M2 can promote tissue repair and regeneration [104]. Injection of azithromycin to mouse models of ischemic stroke induces macrophage polarization from M1 to M2 and reduced ischemic damage in the brain [105]. In a mouse model of muscle injury, injection of gold nanoparticles that deliver IL-4 elevated the number of pro-regenerative M2 macrophages and promoted muscle repair [106]. Injection of (2′Z,3′E)-6-Bromoindirubin-3′-oxime to myocardial infarction rats resulted in high frequencies of M2 macrophages and relieved myocardial infarction symptoms [107]. There have been several approaches to transplant M2 macrophage to treat various diseases. For example, adoptive transfer of M2 macrophages resulted in recovery of cardiac functions in mice with doxorubicin-induced heart failure [108]. In addition, local injection of M2 macrophages to damaged tissue in mouse models of periodontitis could alleviate symptoms of periodontitis [109].

NK cell- and B cell-based immunotherapy

NK cells are known to have conflicting effects on tissue repair. Both depletion of NK cells [21, 22] and activation of NK cells [23] have been reported to support tissue repair. Additionally, injection of antibodies that enhance NK cell activity lead to amelioration of collagen-induced arthritis via suppression of pathogenic follicular T helper cells and Th17 cells and Th17 cells [110]. Furthermore, adoptive transfer of NK cells to EAE mouse models induced in vivo expansion of Treg cells via IL-2 and effectively reduced EAE symptoms [111].

B cell both positively and negatively affect tissue repair. It was demonstrated that treatment of multiple sclerosis patients with rituximab lead to depletion of B cells and reduced inflammatory brain lesions [112]. In contrast, another study has reported that injection of B cells could assist tissue repair through release of regenerative cytokines [57].

Biomaterial-based immunomodulation

As mentioned earlier, various immune cells, such as macrophages, neutrophils, and T cells are involved in tissue repair. They contribute to the removal of pathogens and cellular debris and induce the proliferation and differentiation of stem/progenitor cells. However, excess recruitment and activation can cause chronic inflammation and pathological degenerative diseases. Therefore, strategies for protecting tissues from destructive immune responses and redirecting them toward a pro-regenerative environment are being suggested. In the following sections, therapeutic approaches for tissue regeneration will be discussed in the aspect of immune modulation by delivering recombinant proteins or antagonists for pro/anti-inflammatory cytokines and nanoparticles such as liposomes, polymeric NPs, and extracellular vesicles (EVs) (Table 1).

Table 1.

Summary of tissue regeneration and repair via immune-modulating biomaterials

Classification	Biomaterial components	Application	Results	References
Pro-inflammatory response modulation	Infliximab (anti-TNF-α antibody)	Muscle regeneration	Improvement of muscle weight, myofiber diameter and muscle tissue fraction	[116]
	Glucose-sensitive scaffold loaded with anti-TNF-α antibody	Bone regeneration	Reduction of inflammation. Induction of osteogenesis	[117]
	Gold nanoparticle functionalized with deoxyribozyme	Heart repair	Nanoparticles taken up by macrophage and myocytes. Reduction of TNF-α level and inflammation. Restoration of cardiac function	[118]
	Collagen-hydroxyapatite scaffold loaded with PEI-pDNA nanoparticles encoding IL-1Ra	Bone repair	Inhibition of IL-1β activity. Restoration of osteogenic gene expression and calcium deposition of BM-MSCs	[125]
	TNFR1 antagonist, TNFR2 agonist	Heart repair	Improvement of cardiac function and microglia activity by TNFR2 agonist	[128]
	Hydroxamic acid MMP inhibitor	Neuronal repair	Reduction of vascular permeability. Attenuation of neutrophil infiltration. Induction of locomotor recovery	[130]
	Anti-CCL5 and CXCL4 antibody	Lung repair	Reduction of neutrophil recruitment and CCL5-CXCL4 heteromer formation	[131]
Anti-inflammatory response modulation	Mineral-coated microparticles loaded with IL-10	Neuronal repair	Reduction of pro-inflammatory cytokines. Induction of M2 polarization. Improvement of motor function and axonal restoration	[136]
	IL-10	Muscle regeneration	M2 macrophage polarization, myoblast activation and proliferation	[137]
	IL-4	Neuronal repair	Reduction of inflammation. Elevation of neuronal and oligodendrocyte marker	[141]
	IL-4 complex	Heart repair	Accumulation of M2 macrophage in injured tissue. Reduction of infarct size. Enhancement of cardiac tissue properties	[142]
Immune tolerance induction	Liposome loaded with infarction-specific antigens and rapamycin	Heart repair	Induction of tDC, Treg and M2 macrophage polarization. Improvement of cardiac function	[147]
	PLG nanoparticle loaded with CD4-encoded Dby epitope	Bone marrow transplantation	Induction of long term survival of bone marrow grafts	[148]
Nanoparticle	Negatively charged immune-modifying microparticles	Heart and kidney repair, autoimmune disease	Infusion to inflammatory monocyte. Induction of apoptosis upon spleen sequestration	[153]
	PLG nanoparticle	Neuronal repair	Reduction of neutrophil and monocyte recruitment. M2 macrophage polarization. Improvement of axonal regrowth and locomotor function	[154]
	Allylamine or acrylic acid plasma polymer film, immobilized with gold nanoparticles	Bone regeneration	M2 macrophage polarization, osteogenic differentiation of BMSC	[156]
EV	EV from Treg cell	Neuronal repair	Reduction of inflammation	[159]
	EV from adipose-derived stem cell	Wound healing	Activation of HUVEC. Induction of angiogenesis and collagen deposition	[161]
	EV from induced pluripotent stem cell-derived cardiovascular progenitor	Heart repair	Cardiac cell survival, proliferation and endothelial cell migration. Improvement of cardiac function	[162]
ROS scavenger	Hydrogel composed of ROS cleavable hyperbranched polymer and methacrylate hyaluronic acid, loaded with catalase	Heart repair	Reduction of excess ROS, inflammation and apoptosis. M2 macrophage polarization and angiogenesis. Improvement of cardiac function	[172]
	dMn3O4 loaded PEG-TK-C18 nanomicelles	Kidney repair	Reduction of excess ROS, inflammation, apoptosis and fibrosis. Reduction of tissue damage. Improvement of kidney function	[181]
	Mn doped Co3O4 nanocrystalline	Bone regeneration	MSC adhesion, spreading, proliferation and osteogenic differentiation	[174]
Biomimetic scaffold	Decellularized bone matrix scaffold coated by graphene oxide nanoscrolls wrapping magnesium nanoparticles	Bone regeneration	Recruitment of BMSCs and EPCs. M2 macrophages polarization, angiogenesis and osteogenesis. Improvement of blood vessel and bone formation	[176]
	Mesoporous bioglass scaffold loading dexamethasone and recombinant human bone morphogenetic protein	Bone regeneration	Induction of anti-inflammatory response and M2 macrophage polarization. MSC recruitment and differentiation. Cartilage formation, endochondral ossification and ectopic bone formation	[177]
	High-stiffness transglutaminase cross-linked gelatins, IL-4 and/or SDF-1α incorporation	Bone regeneration	Osteogenic differentiation of BMSCs, M2 macrophage polarization and BMSC recruitment. Induction of periodontal tissue and bone regeneration	[178, 179]
	Silk fibroin-chitin composite nanofiber scaffold loaded with TGF-β1	Cartilage repair	Chondrocyte adhesion, proliferation and MSCs recruitment. Induction of ECM deposition and chondrogenesis. Improved Cartilaginous defect repair	[180]

Delivery of pro- and anti-inflammatory factors

Modulation of the proinflammatory response

TNF-α, a representative proinflammatory cytokine, can activate several immune cells against infection and increase vascular permeability [113]. TNF-α can also deliver a death signals to inappropriate cells such as tumors and, if kept at low levels, participate in injured and senescent tissue remodeling [114]. However, excessive TNF-α can cause autoimmune and degenerative diseases such as rheumatoid arthritis, psoriasis, spinal cord injury, and bone destruction [115]. Therefore, blocking TNF-α signaling has been suggested as a therapeutic strategy and many TNF antagonists, such as infliximab, etanercept, adalimumab (Humira), and certolizumab pegol are approved by the FDA and used in patients mainly with autoimmune diseases. As it has been revealed that TNF-α is also involved in degenerative diseases, studies applying these strategies for tissue regeneration have been widely investigated. Stratos et al. administered infliximab intraperitoneally to rats after a standardized open muscle injury model and observed several signs of skeletal muscle regeneration such as an increased muscle weight ratio, myofiber diameter and muscle force in addition to a reduction in TNF-α in the injured site [116]. Wang et al. focused on severe alveolar bone destruction by TNF-α in diabetic patients and developed a novel glucose-sensitive scaffold for enhanced delivery of TNF-α antibody [117]. After surgery for bone defect construction in a diabetic rat model, the drug-loaded scaffold-implanted group showed reduced inflammation-related protein (TNF-α, CCL2, CXCL1, and NF-κB) expression and increased osteogenesis-related protein expression together with bone repair, as analyzed histologically. Somasuntharam et al. chose an in vivo knockdown strategy using gold nanoparticles (AuNPs) functionalized with deoxyribozyme (DNAzyme), which catalytically silences TNF-α (Fig. 4) [118]. They found enhanced DNAzyme delivery to macrophages in comparison with lipofectamine, and upon intramyocardial administration in a MI model, a significant decrease in inflammation and restoration of cardiac function were observed.

Fig. 4.

A Schematic showing DNAzyme gold particles with three types of particles. Active (Dz), inactive (i-Dz) and nonspecific (Dz-NS) DNAzyme. TNF-α expression levels B when macrophages were treated with each group at 24 h in vitro and C when injected into a rat model of MI at 3 days. Restoration in cardiac function determined through analysis of D fractional shortening and € ejection fraction. Reprinted with permission from ref [118].

The IL-1 family is closely related to the innate immune response and to inflammation [119]. Each IL-1 family member has distinct biological functions, inducing a pro/anti-inflammatory response or acting as a receptor antagonist against each other. IL-1β, the most investigated IL-1 family member, is activated and released by specific proteolysis processes via the NLRP3 inflammasome and mediates the proinflammatory immune response by regulating the recruitment, activation and maturation of several immune cells such as neutrophils, monocytes, T cells, B cells, and DCs [119, 120]. Therefore, inappropriate action of IL-1β is associated with many inflammation-related neurodegenerative diseases, intervertebral disc degeneration, autoimmune diseases, and metabolic syndromes [121–123]. For therapeutic approaches, strategies blocking IL-1 signaling have been widely investigated. IL-1 receptor antagonist (IL-1Ra) is a natural biomolecule blocking IL-1α/IL-1β signaling and has a central role in the immunosuppressive mechanism of mesenchymal stem cells (MSCs) [124]. In an IL-1Ra-dependent manner, MSCs suppress the proinflammatory role of macrophages and DCs, polarize macrophages into anti-inflammatory M2 macrophages, and promote Treg-cell expansion. Thus, MSCs can be a novel cell therapy for treating liver and joint inflammation, lung diseases, and promoting wound healing. Gene delivery encoding IL-1Ra can also increase the therapeutic effect of MSCs for bone regeneration [125]. Furthermore, the recombinant human intrinsic IL-1Ra anakinra was approved by the US Food and Drug Administration (FDA) and showed great effects in various immune-related diseases without serious adverse effects [121]. Other therapeutic biomolecules in the form of soluble decoy receptors and neutralizing monoclonal antibodies have also been widely investigated and used in clinical applications.

However, blocking proinflammatory factors is not a simple therapeutic strategy for tissue regeneration. TNF-α, for example, has multiple biological effects depending on what receptor it binds to. TNFR1 is expressed on almost every cell type and activates two different signaling pathways that either stimulates cell survival and proinflammatory responses, or stimulates apoptosis and cell death [113]. In contrast, TNFR2 is expressed on immune and epithelial cells, and TNFR2 signaling mainly contributes to immune cell activation, migration, and proliferation. Immune suppressive cells also have TNFR2 so that TNF-α can act as an anti-inflammatory cytokine. For example, Yang et al. showed that TNFR2 signaling upon binding to recombinant mouse TNF-α promoted differentiation and proliferation and enhanced the immune suppressive function of Treg cells in vivo and in vitro [126]. TNF-α/TNFR2 signaling may also contribute to the immunomodulatory role of MSCs. Beldi et al. demonstrated that MSCs, compared to TNFR2 knockout, significantly downregulated T-cell proliferation, activation, proinflammatory cytokine production, and enhanced Treg induction [127]. Based on these properties of TNF-α, Gouweleeuw et al. compared selective TNF receptor targeting treatment to nonspecific TNF inhibition for various tissue regeneration situations [128]. They showed that TNFR2 agonists have beneficial effects over TNFR1 antagonists and nonspecific TNF inhibitors in some conditions, such as enhancing cardiac outcome in an MI model and microglial activity in the brain.

Another strategy for modulating the proinflammatory response is to suppress immune cell infiltration into inflamed tissue. In general, MMPs and chemokines are involved in blood vessel permeability and recruitment of immune cells such as neutrophils, monocytes, NK cells, and T cells [129]. Therefore, blocking strategies against these immunomodulants are being studied for tissue regeneration. For example, MMP-9 increases rapidly at the early stage of moderate contusion spinal cord injury [130]. After treatment with an MMP inhibitor, a significant reduction in neutrophil infiltration, reduced damage to the BSC barrier, and locomotor recovery were observed. Acute lung injury after infection is also related to neutrophil recruitment mediated by platelet- and platelet-derived chemokines, specifically CCL5 and CXCL4, and their heteromerization. [131]. Antibodies against CCL5 and CXCL4 reduced neutrophil recruitment and disturbed CCL5-CXCL4 heteromer formation to contribute to lung recovery in various ALI situations. Recruitment of T cells may also contribute to inflammatory diseases, and the chemokine receptor CXCR3 plays a key role in activated T-cell migration. Whereas CXCR3 antagonists have beneficial effects in inhibiting T-cell migration, O’Boyle et al. demonstrated that CXCR3 agonists induce internalization of CXCR3 and other chemokine receptors so that CXCR3 agonists have better function in controlling T-cell migration toward synovial fluid from rheumatoid arthritis patients, which contains multiple chemokines [132].

However, similar to that of the cases seen above, some proinflammatory factors are also involved in pro-regenerative responses. Stromal-derived factor-1 (SDF-1, CXCL12) binds to its receptor CXCR4 and is a well-known chemokine contributing to inflammation, tissue development and tumor metastasis [133]. However, immunosuppressive MSCs also express CXCR4, so SDF-1 delivery can be an effective strategy for tendon, cardiac muscle, and liver regeneration [134]. Based on the polymorphism and multifunctionality of various proinflammatory factors, it is important to identify definite mechanisms of such diseases and apply precisely targeted strategies.

Activation and polarization toward the anti-inflammatory response

IL-10 is an anti-inflammatory cytokine that downregulates proinflammatory cytokine responses, reduces phagocytosis and antigen presentation, and enhances the inhibitory, tolerance, and scavenger effects of immune cells such as M2 macrophages and Treg cells [135]. Hellenbrand et al. intramedullary injected mineral-coated microparticles bound with IL-10 for sustained IL-10 delivery in a spinal cord injury model and observed significant changes in cytokine and macrophage phenotypes [136]. Motor function and axonal restoration in the rubrospinal and reticulospinal tracts were improved so that a novel IL-10 delivery strategy could overcome the blood–spinal cord barrier and have therapeutic effects on neurological disorders. Dysfunction of IL-10 can lead to several infectious and autoimmune diseases. Therefore, many therapeutic applications of IL-10 for tissue regeneration have been investigated. In a rodent hindlimb unloading and reloading model, Deng et al. found that whereas a shift of cytokine responses from Th1 to Th2 is induced during muscle regeneration, M2 macrophage polarization and muscle fiber repair is decreased in a null mutation of IL-10 [137]. They also demonstrated that direct application of IL-10 did not induce myoblast proliferation or MyoD and myogenin expression. In contrast, IL-10-stimulated M2 macrophages had beneficial effects on myoblast activation and proliferation, implying that M2 macrophages are key mediators of IL-10-mediated tissue regeneration. Due to the relationship between inflammation and fibrogenesis, IL-10 treatment can be applied to various fibrotic diseases such as skin, heart, lung, liver, and kidney diseases [138]. Moreover, IL-10 contributes to bone regeneration whilst blocking osteoclast formation and promoting osteoblast differentiation [139].

IL-4, together with IL-13, is known to activate T cell differentiation from naïve T cells to Th2 cells or induce B cell class switching to produce IgE [140]. IL-4 maintains a balance between type 2 immune responses and type 1 immune responses, and if it is excessively expressed, it can cause allergic diseases such as atopic dermatitis, rhinitis, and asthma. IL-4 can also contribute to anti-inflammatory responses and tissue regeneration by polarizing macrophages from the M1 to M2 phenotype and by activating nonhematopoietic cells. Lima et al. [141] demonstrated that intraperitoneal administration of recombinant IL-4 has a neuroprotective role in a spinal cord injury model. Upon administration, they observed a reduction in inflammatory markers (CD11b/c and iNOS) and an elevation in the neuronal marker βIII-tubulin and the oligodendrocyte marker O4 without affecting astrocytes. Shintani et al. administered an IL-4 complex that combined recombinant IL-4 with an anti-IL-4 monoclonal antibody and showed a therapeutic effect on MI [142]. In the injured myocardium, more M2 macrophages accumulate highly expressing genes related to anti-inflammation, angiogenesis, and connective tissue formation. From M2 macrophage deficient Trib1^−/− mice, they demonstrated that therapeutic effect of IL-4 in post-MI model is mediated by its ability to induce M2 macrophages, and not through a direct effect on cardiac cells. The direct effect of IL-4 on nonhematopoietic cells can be important in some cases. Sharon Goh et al. demonstrated that IL-4 secreted from eosinophils is critical in toxin-mediated liver injury repair, not through IL-4Rα signaling in macrophages, but through hepatocytes leading to hepatocyte proliferation [143]. IL-4 can also activate fibroblasts via STAT-6 signaling to produce collagens, which may contribute to ECM formation [144].

In addition to simply administering anti-inflammatory cytokines, immune tolerance induction is considered a novel therapeutic strategy for resolving inflammatory diseases and tissue regeneration in terms of their target specificity without affecting normal immune functions and long-term effects. To mimic the peripheral immune tolerance mechanism, antigens together with tolerogenic modulatory agents are delivered to DCs to induce tolerogenic DCs (tDCs), or peptide-MHC complexes are delivered directly to T cells to induce Treg cells [145]. Various tolerogenic agents such as rapamycin, dexamethasone, vitamin D, IL-10, and TGF-β have been used to induce tDCs. In the case of antigens, antigenic peptides known to be expressed in specific cells or, if not, components of such cells have been used. Maldonado et al. loaded antigens and rapamycin into biodegradable poly(lactide-coglycolide) (PLGA) and demonstrated the induction of antigen-specific Treg cells in vivo and in vitro and its ability to reduce humoral immunity [146]. They also showed therapeutic effects of tolerogenic nanoparticles in animal models such as hypersensitivity, experimental autoimmune encephalomyelitis, and hemophilia. Kwon et al. loaded MI-specific antigens with rapamycin into liposomes (L-Ag/R) and conducted intradermal administration into an MI model [147]. They demonstrated that tDCs followed by Treg and M2 macrophages were well induced by L-Ag/R and that cardiac remodeling and functional resolution were improved. Hlavaty et al. demonstrated tolerance induction by delivering the CD4 epitope Dby rather than the CD8 epitope Uty loaded on the surface or into PLG nanoparticles in a bone marrow transplantation model [148]. According to Clemente-Casares et al., antigenic peptide-loaded pMHC II-attached nanopchcapable? of inducing antigen-specific regulatory CD4 + T-cell type I (T_R1)-like cells which had therapeutic effects on autoimmune diseases such as T1D and EAE models [149]. They also demonstrated that (T_R1)-like CD4 + T cells can only be induced from antigen-experienced T cells, not naïve T cells.

Nanoparticle-based immunomodulation

Various nanoparticles are being used for delivering bioactive molecules such as small molecular drugs, peptides, proteins, DNA, mRNAs, and miRNAs. By loading these molecules into nanoparticles and functionalizing the nanoparticle surface, it is possible to efficiently deliver drugs to the target site while avoiding exposure to the outer environment and increasing the local concentration. It may also be possible to enhance the effectiveness of the drug and minimize side effects by controlling drug release. However, regardless of the loading of bioactive molecules, the physicochemical properties of nanoparticles themselves can affect immunity. Size, shape, stiffness, topography, surface charge, and hydrophilicity are general properties that determine protein corona formation, cellular interaction, toxicity, and downstream signaling of nanoparticles [150]. For example, cationic nanoparticles tend to be more absorbed by DCs in clathrin-mediated processes and effectively stimulate Th1 responses [151]. They can also influence mitochondria and the endoplasmic reticulum so that more ROS and proinflammatory cytokines are released. Although cationic nanoparticles may act as adjuvants, they also possess toxicity to the cell itself and the neighboring environment. Some pH-sensitive cationic liposomes have beneficial effects on endosomal escape, and many clinically approved lipid nanoparticles take advantage of these properties [152]. On the other hand, anionic nanoparticles have little toxicity, and highly negatively charged nanoparticles can bind to scavenger receptors such as macrophage receptor with collagenous structure (MARCO) on immune cells [151]. After being taken up by circulating monocytes and neutrophils, they reprogram immune cells to modulate migration toward lymphoid organs and inflamed tissue. Getts et al. demonstrated that anionic immune-modifying microparticles have therapeutic effects on several inflammation-related diseases such as MI, autoimmune disease, and colitis by regulating circulating monocyte behavior [153]. Shea et al. [154] administered poly(lactide-coglycolide) (PLG)-based nanoparticles with a diameter of 500 nm and a negative zeta potential (< -30 mV) intravenously after spinal cord injury in mice. They observed not only a reduced infiltration of circulating neutrophils and monocytes but also the development of a pro-regenerative environment, defined by M2 macrophage polarization and an increase in the anti-inflammatory markers arginase 1, CD206, and IL-10. By analyzing scar formation, axonal regrowth and locomotor function, they confirmed the therapeutic effect on spinal cord injury soley through the use of a polymer. The hydrophobicity of biomaterials may also modulate the immune response in terms of monocyte adhesion, foreign body giant cell (FBGC) formation, and time-dependent cytokine release [155]. Whereas hydrophobic or hydrophilic/ionic polymeric surfaces are more prone to form FBGCs, hydrophilic/neutral polymeric surfaces activate macrophages and promote the release of cytokines and chemokines such as IL-1β, IL-6, IL-10, IL-8, and MIP-1β. Chen et al. changed the scale of the nanotopography (from uncoated to heights of 1.6, 38, and 68 nm) and analyzed macrophage behavior, the expression of inflammatory cytokines, and osteoclastic, osteogenic, angiogenic, and fibrogenic factors [156]. They demonstrated that 68 nm nanotopography significantly activated macrophages to induce an osteoimmune environment leading to osteogenic differentiation of bone marrow stromal cells.

EV-based immunomodulation

EVs are small vesicles released from cells that contain phospholipid bilayers, membrane and cytosolic proteins, miRNAs, and mRNAs [157]. Based on the property that EVs carry considerable biomolecules from parental cells, many studies have investigated replacing cell therapy with EVs derived from such cells. MSC-derived EVs (MSC-EVs) are one of the most studied regenerative EVs and inherit immunosuppressive roles from MSCs. Different kinds of natural or engineered MSC-EVs have already been proven to have beneficial effects on liver, heart, nervous, bone, cartilage, and wound regeneration [158]. EVs derived from Treg cells (Treg-EVs) can downregulate the inflammatory response. They contain key immunosuppressive molecules characteristic of Tregs, such as IL-35, CTLA-4, and CD73, and can suppress T-cell proliferation and proinflammatory cytokine secretion and induce Tregs in vitro [159]. They also showed beneficial effects on neuroinflammation, autoimmune diseases, and organ transplantation [160]. Liu et al. [161] demonstrated that EVs derived from human adipose-derived stem cells can activate PI3K-AKT-mTOR-HIF-1α signaling in advanced glycosylation end product-treated human umbilical vein endothelial cells (HUVECs). These EVs contributed to angiogenesis and collagen deposition, leading to wound healing. Although EVs mimic parental cell functions, EVs could have unique effects of their own due to their ability to transfer miRNAs to target cells. miRNA is a small, noncoding RNA that binds to mRNA and inhibits its posttranscriptional processes, leading to gene silencing. Harane et al. found that induced pluripotent stem cell-derived cardiovascular progenitor secreting EVs (iPSC-Pg-EVs) contain several miRNAs known to be present in heart and cardiovascular cells and have beneficial effects in heart failure [162]. They demonstrated that iPSC-Pg-EVs enhanced cardiac cell survival, angiogenesis and cardiomyocyte proliferation in vitro, increased cardioprotective genes, and improved cardiac function in a chronic heart failure model.

Another advantage of EVs is known as a ‘safe drug delivery system’, which implies better biodistribution for targeting effects and immune evasion [157]. Although it has been proven that EVs do not show significant immunogenicity and toxicity, these properties depend on whether EVs contain antigens, immunomodulatory factors and routes of administration [163]. When EVs are derived from antigen-presenting cells or target cells themselves, T-cell receptors can directly interact with antigen-loaded MHC molecules present on EVs. Likewise, DC uptake of such EVs can induce an antigen-specific immune response. The biodistribution of EV is somewhat controversial however [157]. Whereas EVs have targeting effects due to surface adhesion molecules, the major organs where EVs accumulate are the liver, lung, and kidney. Therefore, further studies need to be performed to engineer EVs and apply them to other biomaterials.

ROS scavenger-based immunomodulation

ROS are highly reactive chemical groups derived from oxygen molecules and are generated by NADPH oxidase-mediated or mitochondria-mediated mechanisms [164, 165]. Due to their ability to oxidize proteins, lipids and nucleic acids, ROS can directly attack pathogens and contribute to host defense, which is termed as a ‘respiratory burst’. In cellular signaling, ROS enhance proinflammatory innate responses in terms of inflammasome formation and antiviral responses. Further studies revealed that ROS can affect several cell behaviors, such as proliferation, differentiation, activation, senescence, and apoptosis. When it becomes excessive, ROS create oxidative stress leading to chronic inflammation and pathogenesis, such as multiple sclerosis, rheumatoid arthritis, and renal and gastrointestinal mucosal diseases [166–169].

ROS scavengers are molecules that can react with ROS and resolve the oxidative environment created by ROS. Various catalytic enzymes such as catalase, peroxidase, superoxide dismutase, and nanozymes, which are composed of inorganic and polymeric nanomaterials mimicking enzymatic functions, are being investigated for tissue regeneration [170, 171]. Ding et al. [172] developed a multifunctional hydrogel that contains an ROS-scavenging hyperbranched polymer and oxygen-generating catalase (Fig. 5). Upon administration into the infarcted rat heart, they observed reduction of apoptosis, inflammation and infarcted size together with enhanced myocardial function and vascularization. Choi et al. investigated a novel delivery system for 1-dodecanethiol-stabilized Mn₃O₄ (dMn₃O₄) as an inorganic nanozyme [173]. dMn₃O₄ was loaded into PTC nanomicelles that contained an ROS reactive thioketal linker so that nanozymes were selectively released into the ROS-rich inflamed tissue. By administrating these PTC-M nanoparticles into the tail vein, they showed therapeutic effects in an ischemia reperfusion-induced acute kidney injury model. Tian et al. demonstrated the stem cell regulatory effect of nanozymes [174]. They fabricated nanocrystalline Co₃O₄ by doping Mn so that an increased ratio of Co²⁺/Co³⁺ significantly improved the ROS scavenging effect. By treating human MSCs, cellular survival, adhesion, spreading and proliferation were enhanced, and osteogenic differentiation was resumed. This implies the possibility of using ROS scavengers as a synergistic strategy with other tissue regenerative therapies.

Fig. 5.

Mechanism of multifunctional hydrogels for ROS scavenging and oxygen generation. Reprinted with permission from ref [172].

Biomimetic scaffold-based immunomodulation

The basic concept of scaffolds for tissue regeneration or repair is to play the roles of the natural ECM during the process of tissue regeneration [175]. As the importance of immune system in the tissue regeneration process is becoming more apparent, scaffolds have been investigated in greater depth as a tool for immunomodulation. For this purpose, scaffolds can be modified in terms of bioactive molecule loading and its physicochemical properties. For bone regeneration, Zheng et al. developed decellularized bone matrix scaffold coated with magnesium-enriched graphene oxide (GO) nanoscrolls (MgNPs@GNS/DBM) [176]. In the initial stage of the tissue repair, GO in MgNPs@GNS/DBM activated pro-inflammatory M1 macrophages in both phagocytic and TLR4 binding pathways, and recruited bone marrow-derived MSCs (BMSCs) and endothelial progenitor cells (EPCs) for bone repair. In the later stage of the tissue repairs, magnesium in MgNPs@GNS/DBM decreased GO-induced inflammation by blocking NF-κB signaling and repolarized M1 macrophages to anti-inflammatory M2 macrophages. By balancing pro-inflammatory and anti-inflammatory responses, GO coupled with magnesium exerted angiogenic and osteogenic effects in vitro. Upon implanting biomimetic MgNPs@GNS/DBM scaffolds to cranial bone defects of animals, significant bone regeneration was observed.

Another approach for bone regeneration is to modulate inflammation in bone formation steps of endochondral ossification (EO) [177]. Hierarchical mesoporous bioglass scaffolds, which are widely used as biomimetic bone substitute, were loaded with dexamethasone and recombinant bone morphogenetic protein (rBMP) for immunomodulation. Dexamethasone was released quickly in the early stage of EO process and facilitated anti-inflammatory response by inhibiting excessive macrophage recruitment and inducing M2 macrophages. Meanwhile, rBMP was released slowly up until the later stage of EO and contributed to mesenchymal condensation and chondrocyte activation. The spatiotemporal release of both molecules in the biomimetic scaffolds enabled significant endochondral ossification and ectopic bone formation.

Physicochemical properties of biomimetic scaffold are important factors in scaffold-based immunomodulation. To mimic the environment of bone ECM, He et al. developed high-stiffness transglutaminase cross-linked gelatin (TG-gel) by controlling the gelatin concentration [178]. Upon encapsulating BMSCs into TG-gel, mineral deposition and expression level of osteogenesis-related genes increased as the stiffness increased. However, macrophages in high-stiffness TG-gel tended to polarize toward the M1 phenotype and had adverse effects on osteogenic differentiation of BMSCs. To improve the hydrogels, IL-4 or SDF-1α was loaded in the hydrogels for macrophage repolarization and cellular recruitment [179]. Upon implanting drug-loaded high-stiff TG-gel to periodontal defect animal model, CCR7⁺ M1 macrophages decreased in the defect region while CD206⁺ M2 macrophages and CD44⁺ BMSCs increased. As a result, enhanced periodontal tissue regeneration was observed in the drug-loaded hydrogels.

Cartilage regeneration has been achieved using silk fibroin (SF)-chitin composite nanofiber scaffold loaded with TGF-β1 [180]. SF-chitin nanofibers mimic the topological structure of type II collagen in cartilage and enhanced chondrocyte adhesion and proliferation due to morphological changes in the chondrocytes. TGF-β1 released from the scaffold stimulated migration and differentiation of MSCs. Scaffold implantation to cartilage defect of animal model resulted in complete filling with cartilaginous tissue.

Conclusions

Various immune cells participate in the tissue repair process. These cells modulate the tissue repair processes of inflammation, anti-inflammation, repair, and finally tissue regeneration. Neutrophils, macrophages, and T cells, and other immune cells have multifaceted and at times opposing roles and phenotypes that can induce or inhibit inflammation. Thus, a deeper understanding of immune cells is needed to properly modulate immune cells in the tissue repair processes.

Treg cells, a key modulator of tissue regeneration and repair, interacts with various immune cells such as macrophages, DCs, neutrophils, NK cells, and other types of T cells to downregulate the immune response and control inflammation. Treg cells can be used in the treatment of various diseases including autoimmune diseases such as RA and systemic lupus and inflammatory diseases such as MI, acute lung injury, and inflammatory bowel disease. To treat these diseases via Treg cells, both cell therapy and drug therapy can be used. Cell therapy, which transfers ex-vivo expanded Treg cells to patients, needs to achieve both antigen specificity sufficiency in cell quantity. Unlike polyclonal T cells that are not antigen-specific, engineered TCR and CAR technologies can attain both antigen specificity and abundant yield of Treg cells. However, these therapies still need improvements to treat diseases effectively.

Biomaterials can be used to modulate immune response in vivo for tissue regeneration. Biomaterial-mediated delivery of bioactive molecules has been widely investigated to downregulate destructive immune response and promote pro-regenerative responses. Tissue repair with drug delivery can be further improved by modulation of physicochemical properties of carrier nanoparticles, application of environment-sensitive functional polymers, and engineering of cell-derived EVs as delivery carrier.

Although recently developed immune modulating therapeutics have shown considerable therapeutic effects, they still exhibit unwanted off-target effects and suboptimal biodistribution. More effective immune modulation strategies will be developed as the precise mechanisms underlying pathogenesis and the roles of immune cells in tissue injury are elucidated.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Ethical statement

There are no animal experiments carried out for this article.