Engineering of Immune Microenvironment for Enhanced Tissue Remodeling

Abstract

The capability to restore the structure and function of tissues damaged by fatal diseases and trauma is essential for living organisms. Various tissue engineering approaches have been applied in lesions to enhance tissue regeneration after injuries and diseases in living organisms. However, unforeseen immune reactions by the treatments interfere with successful healing and reduce the therapeutic efficacy of the strategies. The immune system is known to play essential roles in the regulation of the microenvironment and recruitment of cells that directly or indirectly participate in tissue remodeling in defects. Accordingly, regenerative immune engineering has emerged as a novel approach toward efficiently inducing regeneration using engineering techniques that modulate the immune system. It is aimed at providing a favorable immune microenvironment based on the controlled balance between pro-inflammation and anti-inflammation. In this review, we introduce recent developments in immune engineering therapeutics based on various cell types and biomaterials. These developments could potentially overcome the therapeutic limitations of tissue remodeling.

Introduction

Tissues have a self-regenerative capacity to recover damaged constructs and their functionalities [1]. Various tissue engineering approaches including functional biomaterials [2], drug-eluting systems [3], and stem cell therapeutics [4] have been applied to enhance tissue regeneration. However, unexpected immunomodulation after the treatment could reduce the effectiveness of the technologies applied and form fibrotic tissues, which in turn reduces therapeutic results and causes prolonged side effects. Recently, the crosstalk between lesions and the immune system has been highlighted as an important mediator for improving the outcome of tissue regeneration [1, 5]. Immune cells actively participate in the following steps of tissue remodeling: inflammation, new tissue formation, and maturation [6]. In the initial stage, immune cells (e.g., neutrophils, NK cells, and M1 macrophages) are recruited to the wound site for inflammation. In addition, antigens and debris are removed to protect the wound from foreign materials. Subsequently, pro-regenerative immune cells (e.g., M2 macrophages) collaborate with fibroblasts to construct foundations for tissue remodeling by synthesizing multiple cytokines, promoting extracellular matrix formation, and recruiting stem cells in the damaged tissue. The combination of immunological factors (including cells, chemokines, and mechanical cues), composing the microenvironment during whole process of tissue remodeling is referred as immune microenvironment. Immune responses during tissue remodeling are complex and dynamic. These induce both pro-inflammatory and anti-inflammatory reactions. Thus, alteration of the duration and inflammatory balance at each immunological step can substantially affect regeneration efficacy.

As a convergence of immunology and regenerative medicine, various engineering tools and fabrication techniques have been applied for efficient immunomodulation and controlled tissue remodeling [7, 8]. Recently, cellular engineering, functional scaffold systems, and targeted chemokine delivery techniques for immune system control have yielded successful results in the regeneration of damaged tissues. Regenerative immune engineering typically involves the control of immune cells and cytokine secretion by the participating cells through the provision of a favorable microenvironment for the desired immune responses and subsequent tissue regeneration (Fig. 1). Here, we review recent artworks of regenerative immune engineering and describe various cells, biomaterials, and drug delivery techniques for enhanced immunomodulation and tissue regeneration.

Fig. 1.

Schematic illustration of immune microenvironment and its role in tissue remodeling at the lesion. Line arrows indicate cellular migration into the lesion. Dashed arrows indicate differentiation of immunocytes. Treg; regulatory T cell, Th2; T helper 2 cell, M1; type-1 macrophage, M2; type-2 macrophage, TGF-β; transforming growth factor-β, IL; Interleukin, IFN-γ; interferon-γ, TNF-α; tumor necrosis factor-α, SAP; serum amyloid P, VEGF; vascular endothelial growth factor

Cells

Different types of immune cells directly or indirectly participate in the wound healing process by secreting various cytokines and inducing pro-inflammation and anti-inflammation. Pro-inflammatory cytokines such as interleukin (IL)-1, IL-6, IL-17, and tumor necrosis factor-α (TNF-α) activate lymphocytes (e.g., T cells and NK cells) and phagocytosis of macrophages to induce inflammatory responses and remove infectious antigens. Meanwhile, representative anti-inflammatory cytokines such as IL-4, IL-10, IL-13, and transforming growth factor-β (TGF-β) induce polarization of macrophages into M2 type and activate type-2 immunity. Thereby, these are involved in triggering anti-inflammation and tissue regeneration. In this section, we describe cell-based regenerative immune engineering approaches using various cell types and the cytokines secreted by these for enhanced tissue regeneration (Table 1).

Table 1.

Various cell types used in cell-based immunomodulation

Cell type	Target tissue	Target disease	Description	Reference
Macrophage	Heart	Myocardial infarction (MI)	IL-101 treatment increases M2 macrophage polarization	[12]
			IL-42 treatment increases M2 macrophages polarization	[13]
			Macrophage transplantation initiates immunomodulation	[25]
		Apical resection (AR)	Macrophage transplantation initiates immunomodulation	[26]
	Lung	Lung fibrosis	SAP3 inhibits M2 macrophage accumulation	[14]
			Microcystin-LR increases M2 macrophages polarization	[19]
			TNF-α4 decreases lung fibrosis	[20]
		Acute lung injury (ALI)	Macrophage transplantation initiates immunomodulation	[28, 29]
	Kidney	Renal fibrosis	MyD88 Signaling Pathway involves a Th2 immunomodulation and M2 Macrophages polarization	[17]
		Acute kidney injury (AKI)	Macrophage transplantation initiates immunomodulation	[27]
	Skin	Pressure ulcers (PUs)	PSLs5 increases M2 macrophage polarization	[21]
	Liver	Drug-induced liver injury (DILI)	CCL5 increases M1 polarization and inhibits M2 polarization	[23]
		Post-hepatectomy, liver dysfunction	M2-BMDM6 infusion increases M2 macrophages polarization and S1P7 generation	[24]
T-cell	Heart	Myocardial infarction (MI)	CD4 + T lymphocyte activation	[35, 36]
			Superagonistic anti-CD28 monoclonal antibody activates Tregs8	[37]
	Muscle	Skeletal muscle injury in aging	IL-339 treatment activates Tregs	[38]
		Muscular dystrophy	IL-210/anti-IL-2 complexes activate Tregs and increased IL-10 concentrations	[39]
	Bone	Osteoporosis	Tregs inhibit the differentiation of osteoclasts, while Th1711 cells promote the differentiation of osteoclasts	[42]
		Skeletal tissue regeneration	γδ T cells produce IL-17A12, which promotes bone formation and facilitates bone fracture healing	[43]
	Brain	Parkinson’s disease	Anti-TNF-α antibody administration and Tregs transfer increase Tregs and reduced Th1 cells	[44]
		Ischemic stroke	Endogenous Tregs boosted by CD28SA13 increase M2 macrophage/microglia	[45]
Stem cell	Intestine	Inflammatory bowel diseases (IBD)	MSC single administration induces long-term regulation of inflammatory response	[50]
			TSG-614 induce M2 macrophage switch	[51]
			MSC15-derived exosomes modulate IL-716	[52]
			MSC-derived exosomes carrying miR-378a-5p17 inhibit NLRP3 inflammasomes	[53]
			miR-200b18-MV19s increase miR-200b and alleviate fibrosis	[64]
		Intra-villi microvascular injury	MSC-derived secretome decrease IL-1α, IL-6 and TNF-α	[59]
	Bone	Rheumatoid arthritis	loss of A20 in MSCs regulate the Th17/Treg balance	[54]
			CXCR720 promotes MSC-mediated immunomodulation	[55]
		Osteoarthritis	MSC-MVs (Nano-ghost) decrease PGE221	[63]
		Blood-spinal cord barrier injury	MSC-EVs attenuate the BSCB22	[67]
	Liver	Acute and chronic liver injuries	CHI3L123 inhibits the proliferation and function of activated T cells	[58]
		Acute liver failure (ALF)	STAT624 activation increases M2 macrophage polarization	[60]
	Kidney	Renal fibrosis	MSC-MVs ameliorate renal inflammation and increase M2 macrophage polarization	[62]
	Lung	Acute respiratory distress syndrome (ARDS)	hEPC25 exosomes deliver miRNA-126 into the injured alveolus	[65]
	Heart	Myocardial infarction	Exo-SDF127 protected activated the PI3K28 signaling pathway	[66]

¹IL-10: Interleukin-10

²IL-4: Interleukin-4

³SAP: Serum amyloid P

⁴TNF-α: tumor necrosis factor-α

⁵PSLs: Phosphatidylserine-containing liposomes

⁶M2-BMDM: Bone marrow derived macrophage

⁷S1P: Sphingosine-1-phosphate

⁸Tregs: Regulatory T cells

⁹IL-33: Interleukin-33

¹⁰IL-2: Interleukin-2

¹¹Th17: T helper 17 cell

¹²IL-17A: Interleukin-17A

¹³CD28SA: CD28 superagonistic monoclonal antibody

¹⁴TSG-6: tumour necrosis factor-α-induced gene/protein 6

¹⁵MSC: Mesenchymal stem cell

¹⁶IL-7: Interleukin-7

¹⁷miR-378a-5p: Micro ribonucleic acid-378a-5p

¹⁸miR-200b: Micro ribonucleic acid-200b

¹⁹MV: Microvesicle

²⁰CXCR7: C-X-C chemokine receptor type 7

²¹PGE2: Prostaglandin E2

²²BSCB: Blood-spinal cord barrier

²³CHI3L1: Chitinase-3-like protein 1

²⁴STAT6: Signal Transducer And Activator Of Transcription 6

²⁵hEPC: Human endothelial progenitor cell

²⁶miRNA-126: Micro ribonucleic acid-126

²⁷Exo-SDF1: Exosome-stromal-derived factor 1

²⁸PI3K: phosphoinositide 3-kinase

Macrophages

Macrophages can be polarized into two phenotypes, type 1 (M1) and type 2 (M2), depending on the microenvironment of the local tissue. Furthermore, each type produces various cytokines to control immune responses. M1 macrophages are conventionally activated by chemokines such as TNF-α, interferon-γ (IFN-γ) and bacterial lipopolysaccharide (LPS) [9]. This results in inflammation, which generally protects our body from infectious materials [10]. Meanwhile, activation of M2 macrophages triggers secretion of anti-inflammatory cytokines, thereby inducing immunosuppression and tissue growth [10]. In the event of an incomplete transition of the macrophage population from M1 to M2, successful tissue remodeling could be retarded, which would result in scar tissue formation and malfunction of the regenerated tissue. Therefore, modulation of the macrophage population during tissue remodeling has been widely studied for enhancing the therapeutic efficacy of wound treatment.

Managing the phenotypes of the macrophage population has been one of the main targets for regenerative immunomodulation. Cardiovascular diseases (CVDs) including myocardial infarction (MI) constitute one of the leading causes of death worldwide [11]. Recent evidences strongly support the concept that cardiac macrophages play crucial roles in the progression and repair of myocardial infarction. In general, healthy tissues can rapidly deploy M2 macrophage-mediated reactions for tissue repair, but tissues with CVD cannot effectively involve a transformation of the macrophage phenotype from M1 to M2 [11]. Therefore, infusion of anti-inflammatory cytokines such as IL-4 and IL-10 into mouse with CVD efficiently induced M2 polarization of macrophages and reduced inflammation in the defect [12, 13]. This, in turn, successfully mitigated fibrosis and improved post-MI cardiac physiology [12, 13].

Although M2 macrophages are involved in pro-regenerative processes, prolonged activation of M2 macrophages accumulating pro-fibrotic factors can cause excessive proliferation of fibroblasts, leading to fibrosis in various organs [14–17]. Pathogenesis of pulmonary fibrosis, which causes severe respiratory disorders, can be promoted by high levels of TGF-β1 secreted from M2 macrophages. This process includes extracellular matrix deposition and differentiation of fibroblasts into myofibroblasts [14]. Therefore, treatment with pro-inflammatory molecules such as serum amyloid P (SAP), recombinant TNF-α, and anti-IL-33 has shown therapeutic effects in mitigating excessive M2 macrophage activation and fibrosis in pulmonary tissue [14, 18–20]. Similarly, suppression of M2 macrophages in other fibrotic tissues of liver, heart, and kidney was also suggested as a novel therapeutic approach in restoring malfunction and damage caused by prolonged M2 macrophage activation [15–17].

Imbalance in M1 and M2 macrophages is also known to be associated with the development of structural and functional tissue deteriorations. Overexpression of M1 macrophage in pressure ulcers (PUs) originated from continuous pressure on skin tissue delays the healing of collapsed tissue and even causes severe inflammation and blood poisoning [21, 22]. Artificially induced M2 polarization by artificial apoptotic cell mimics in a mouse model of PU successfully inhibited ulcer formation and accelerated wound healing, demonstrating its efficacy as a novel therapeutic approach for treating patients at high risk of PU [21]. In addition, the M1/M2 population in liver tissue plays a crucial role in the development and regeneration of hepatic failure as well. Recent studies reported that blocking M1 polarization and infusion of M2 macrophages significantly delayed drug-induced liver injury and alleviated liver tissue dysfunction after liver hepatectomy, respectively [23, 24].

In addition to the modulation of the immune microenvironment for desired macrophage polarization, recent studies have demonstrated the potential of macrophage transplantation to restore damaged tissues. For example, transplantation of chemically induced M2 macrophages efficiently recovered dysfunctions caused by the infarction [25]. The successful engraftment of the transplanted M2 macrophages increased the number of reparative macrophages within the treated tissue and thereby enhanced post-MI regeneration [25]. Another study also attested the therapeutic effects of transplanted macrophages in cardiac recovery by inducing cardiomyocyte proliferation in the MI model of mice [26]. Similarly, injection of M2 macrophages served to reduce inflammatory responses in renal tissues and enhanced proliferation of proximal tubular epithelial cells, which in turn induced healing process after acute kidney injury [27]. Moreover, pulmonary transplantation of macrophages has been highlighted as a novel therapeutic for treating the unregulated macrophage population in lung injuries [28, 29]. Intrapulmonary injection of macrophages greatly reduced the volume of inflamed lung tissue and engineered macrophages secreting IL-4 particularly alleviated inflammation and improved the survival rate of animals after transplantation into acute lung injured mice [28, 29].

These studies indicate that the balance of macrophages between inflammatory and anti-inflammatory reactions is crucial for successful tissue regeneration. Although dynamic interactions with the microenvironment induce persistent phenotypic transformations (rendering it difficult to control), recent studies have achieved significant advances in understanding the underlying mechanisms and exploiting those in damaged tissue restoration.

T-cells

T-cells are one of the most important immune cells in innate and adaptive immunity. It manages disease progression and healing in the body. T helper 1 cell (Th1)-associated type 1 immunity produces pro-inflammatory cytokines such as interferon (IFN)-γ, IL-2, and TNF-α, activating protective inflammation in the body [30] Meanwhile, type 2 immunity, classically perform pathogen-specific adaptive immune responses, generates cytokines such as IL-4, IL-5, IL-10, and IL-13 inhibit excessive type 1 inflammation [30, 31]. Moreover, recent works have discovered a new role of type 2 immunity in regulating functional homeostasis and tissue regeneration [30]. Another type of T-cells participating in type 2 immune responses, regulatory T (Treg)-cells, have also gained substantial attention as potential target cells for enhanced tissue recovery and regeneration. Tregs generally control excessive inflammation in the body, which can even develop into various autoimmune diseases. Although the mechanisms and interactions with other immunocytes remain unclear, recent studies have revealed that Tregs are directly or indirectly involved in tissue healing processes by inhibiting initial inflammatory reactions and regulating neutrophils, macrophages, and T helper cells during regeneration [32].

Modulation of T-cells has shown substantial potential in treating MI. After the injury to the cardiac tissue, innate immune responses are activated first [33]. Then, the adaptive immunity-related CD4⁺ T lymphocytes mediate successful cardiac tissue regeneration [34, 35]. Especially, Treg cells have shown great therapeutic potential in reparative immunomodulation and treatment of MI. Weirather et al. have revealed that depletion of Treg cells increases the number of infiltrated inflammatory immunocytes and deterioration of the infarction, whereas Treg cell activation induces M2 macrophage polarization and remodeling of the cardiac tissue [36]. In another study, the transfer of Treg cells into an MI rat model successfully attenuated inflammatory responses and cardiac dysfunction caused by injury [37]. The anti-inflammatory activity of Treg cells reduces interstitial fibrosis, adverse tissue remodeling, and cardiac apoptosis, resulting in successful cardiac tissue remodeling and restoration of cardiac function [37].

Treg cells are also closely related to the development of skeletal muscle diseases. In general, IL-33 regulates Treg cell homeostasis in injured muscle tissues. However, defects in IL-33 secretion and subsequent Treg cell accumulation at the wound (particularly in aging mice) delay the repair of muscular damage [38]. Treatment with IL-33 and restoration of Treg cell population could facilitate regeneration of acute muscular injury in aging mice. The increased number of Tregs in muscle tissue could alleviate muscular dystrophy such as Duchenne muscular dystrophy (DMD) by improving muscle fibrosis and anti-inflammatory responses with increased IL-10 secretion [39].

In addition, subtypes of T-cells have different roles in maintaining homeostasis and remodeling defects in bone tissue [40, 41]. T helper 17 (Th17) cells are known to promote osteoclast differentiation [42]. However, Treg cells inhibit the differentiation of osteoclasts. Therefore, a population imbalance between Th17 and Treg cells can largely affect the condition of bone homeostasis and can also develop systemic bone diseases such as osteoporosis [42]. Meanwhile, Treg cells induce differentiation of osteoblasts and epithelial gamma delta (γδ) T cells produce IL-17A, a regenerative cytokine, which all can accelerate bone regeneration [42, 43]. Although the underlying modes of action could vary, different subtypes of T-cells mediate successful tissue remodeling in muscle and bone tissues.

Treg cell-based therapies have also gained substantial interest as an immunomodulatory approach for treating brain disorders. T-cells are involved in the development and progression of brain disorders such as Parkinson's disease (PD), a representative neurodegenerative disease. PD patients were observed to have fewer Treg cells and more Th1 cells compared with healthy volunteers. Therefore, the delivery of Treg cells and neutralization of TNF-α has been suggested as a therapeutic approach for reducing the severity of PD [44]. In addition, immunosuppressive Treg cells are downregulated in ischemic stroke patients and this is exacerbated in patients with type 2 diabetes mellitus [45]. Although the dependence of therapeutic outcomes on the severity and dynamics of post-stroke immunity is an area of contention [46], Treg augmentation could modulate immune responses and exhibited potential for reducing the volume of infarction and improving neurological recovery [45].

From the perspective of regenerative immunity, accumulated evidence revealing dynamic transformations in T-cell populations during and after tissue damage indicates the importance of balance between inflammatory T-cells and Tregs for regulating disease development and regeneration of damaged tissues. Many researchers have suggested T-cell-based therapeutics by inhibiting inflammatory reactions and enhancing anti-inflammatory reactions through the modulation of Treg cells. The mechanism of Treg modulation is still unclear owing to the complexity of T-cell-mediated immunity. Nonetheless, it is considered that the provision of a suitable environment for immunomodulation using biomaterials and various engineering techniques could play essential roles in improving the efficacy of T-cell therapies in tissue remodeling and functional restoration of damaged tissue.

Stem cells

Stem cells have been highlighted as the most potential cell source for cell therapy to restore damaged tissues based on their capability to self-renew and differentiate into functional cells [47]. Apart from tissue regeneration, stem cell has demonstrated high efficacy in the treatment of immunological disorders. In particular, mesenchymal stem cells (MSCs), a type of adult stem cell mainly isolated from bone marrow and fat tissue, have been demonstrated to modulate the immune system [48]. MSCs can inhibit innate and adaptive immune responses by producing various immunosuppressive molecules including TGF-β, leukocyte inhibitory factor (LIF), prostaglandin E2 (PGE2), and nitric oxide (NO) [49]. In addition, MSCs suppress type-1 immunity by controlling the proliferation and phenotypic transformations of T-cells, natural killer (NK) cells, dendritic cells (DCs), mast cells, and macrophages [48]. Furthermore, MSCs gain immunosuppressive properties by inflammatory cytokines and subsequently produce various anti-inflammatory substances [48, 49]. This induces the improved activity of Treg cells and transition of macrophages from the M1 to the M2 phenotype [49].

Although most MSC-based therapeutics are aimed at reconstructing damaged tissue through vascularization and remodeling of extracellular matrix (ECM) in the wounds, the immunomodulatory properties of MSCs enable their application in the treatment of immunological disorders. Intestinal bowel disease (IBD) is one of the most prevalent autoimmune diseases caused by persistent inflammation in the gastrointestinal tract. It is accompanied by diarrhea, pain, and surgical resection in severe cases. Alves et al. demonstrated that an MSC administration efficiently reduced the number of Th1 and Th17 cells (which are important in IBD pathogenesis) and enhanced Th2 cell activities and anti-inflammatory cytokine secretion. These, in turn, contributed to pathological improvement with functional restoration of the intestine [50]. The factors secreted by MSCs, including TNF-induced protein 6 and exosomes, could also mediate tissue remodeling in intestinal colitis through M2 macrophage polarization and the production of anti-inflammatory cytokines such as IL-10 [51–53].

MSCs have been demonstrated to have the potential for rheumatoid arthritis (RA), another immune disorder with excessive inflammatory reactions [54]. In patients with RA, the expression level of A20, an inhibitor of inflammatory cytokine secretion, was downregulated significantly. Thus, overexpression of A20 successfully reduced excessive IL-6 production and restored balance between T17 and Treg cells [54]. Especially, increased expression of a specific receptor (C-X-C chemokine receptor type 7 CXCR7) on MSCs has been demonstrated to play multiple roles in regeneration and immunomodulation for arthritis treatment. These include enhanced chondrogenic differentiation, Treg differentiation, and the production of various anti-inflammatory cytokines [55].

MSC-derived cytokines, lipids, and glycoproteins also have been shown therapeutic potential: these enhance anti-inflammatory reactions and healing of hepatic injuries [56–59]. In particular, Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling are crucial in regulating immune reactions in various tissues. MSCs secrete factors that control the JAK-STAT signaling pathways in liver tissue [58, 60]. For example, MSCs inhibit T-cell proliferation and pro-inflammatory cytokine production by suppressing the phosphorylation of STAT1 and STAT3 [58]. In addition, STAT6 activation by MSCs induces M2 macrophage polarization and enhances the expression of anti-inflammatory markers such as CD163, IL-4, TGF-β, and IL-10 [60]. In turn, immune suppression by MSCs through JAK-STAT signaling regulation could alleviate hepatic injuries and was indicated as a potential target for enhanced regeneration through MSC-mediated immunomodulation [58, 60].

MSC-derived vesicles have also emerged as mediators of tissue regeneration. Notwithstanding the great regenerative capacity of MSCs after transplantation, the application of stem cells can cause tumor tissue formation and immune rejection [61, 62]. However, extracellular vesicles (EVs) containing many bioactive molecules secreted from MSCs can circumvent safety issues while enhancing the recovery of injuries in various tissues. In addition, the preserved targeting moiety of stem cells was transferred onto the generated particles and enabled controlled local modulation of the anti-inflammatory environment for cartilage damage [63]. The identification of the molecules that mainly affect regeneration would be complex, but the MSC-derived EVs decreased immune cell migration and pro-inflammatory cytokine secretion, and promoted the shift of macrophages from M1 to M2. Thus, these demonstrated therapeutic outcomes in treating disorders in the liver [62], cartilage [63], intestine [64], lung [65], heart [66], and spinal cord [67]. In addition to regeneration through cellular differentiation, immunomodulation by stem cells plays a crucial role in the balancing of macrophages and T-cells in the immune system, and further improves the efficacy of wound healing.

Future direction of cell-based regenerative immunotherapies

Previous studies have revealed indispensable roles of the immune system in successful tissue regeneration. Various cell types including immunocytes and stem cells contribute reparative immune microenvironment by inducing cellular differentiation and secretion of anti-inflammatory cytokines. Especially, controlling the innate immune cells or injection of those cells have been proved as efficient approaches not only to restore lesions and damages caused by trauma but also to normalize imbalance in immune system in patients. However, despite the complex interplays of immune cells during tissue remodeling, most of the previous studies rely on the evaluation of specific cell types such as macrophages, Tregs, and stem cells, and the contribution of other immune cells have been barely studied. In addition, more comprehensive understanding on what extent of immune modulation would be optimal for successful tissue remodeling need to be further evaluated in future studies. For example, excessive activation of regenerative immune responses can trigger the deposition of ECMs, resulting in fibrotic tissue formation [68]. Therefore, the development of immune cell-based therapeutics with timely strategies and optimized modulation of the immune system would allow successful tissue remodeling with minimal side effects.

Biomaterials

Potential therapeutics that modulate the immune system can be supplemented and improved by functional biomaterials. Although cells can affect the process of immune responses and tissue remodeling by themselves, biomaterials with remarkable biocompatibility and low cytotoxicity have further enhanced cellular viability and long-term functionality for the desired immune responses [7, 69]. Because immunocytes consistently interact with other immune cells and adjacent tissue microenvironments, providing tissue-like substrates that are favorable to regenerative immune cells could induce immune responses for enhanced tissue regeneration [8]. This section reviews various biomaterial-based approaches for successful wound healing by immunomodulation. These approaches include functional hydrogels, tissue-like three-dimensional (3D) scaffolds, and targeted delivery of cytokines using biomaterials (Table 2).

Table 2.

Various biomaterials in immunomodulation

Material type	Target tissue	Used material	Description	Reference
Macroscopic 3D scaffold	Bone	MCPC1	MCPC increases TGF-β12 and decrease TNF-α and IL-63	[72]
		Biphasic calcium phosphate	Dual-wavelength photosensitive scaffold regulates macrophages polarization and inhibits the maturation of dendritic cells	[73]
		Strontium	IFN-γ4/SrBG5 scaffolds regulate M1 and M2 macrophage polarization	[74]
		GelMA, Gelatin, PEG, MSN	BMP-46 from MSNs7 increase M2 macrophage polarization	[86]
Hydrogel	Bone	Akermanite, Alginate	Injectable Aker/SA8 hydrogel increases M2 macrophage polarization	[80]
		Alginate, Sericin, Graphene oxide	Injectable Alg/Ser/GO9 hydrogels increase M2 macrophage polarization	[83]
		Nanosilver, halloysite, GelMA10	Anti-bacterial hybrid hydrogel modulates inflammatory cytokines and enhanced the osteogenic differentiation	[87]
		GelMA, Nanofish bone	GelMA/Nanofish bone hydrogel increases immunomodulation	[88]
		PRP11, GelMA	The 3D-printed PRP-GelMA composite increases M2 macrophage polarization	[90]
	Skin	Bioactive glass, Sodium alginate	BG/SA hydrogel increases M2 macrophage polarization	[79]
		Silk	Induced more pro-regenerative macrophages and recruited more hair follicle stem cells	[81]
		GAG12, PEG13	Scavenge the inflammatory chemokines MCP-114, IL-815, MIP-1α16 and MIP-1β17	[82]
		GelMA, Surfactin A	Promote diabetic wound healing via regulating M2 macrophage polarization and promoting angiogenesis	[89]
Decellularized matrix	Muscle	Heart dECM18	MSC + dECM scaffold generated synergistic effects	[97]
	Spinal cord	Brain dECM	Brain dECM increases M2 macrophage polarization	[98]
	Cartilage	Cartilage dECM	Cartilage dECM scaffolds with IL-4 increase M2 macrophage polarization	[99]
	Ligament, Bone	xNDM19	xNDM which carried the rosiglitazone promote the ligament-to-bone regeneration by PPARγ	[100]
	Kidney	PLGA20 (PME21), Kidney dECM, PDRN22, MSC-EV	PME/PDRN/IFN-γ-primed MSC-EV complex effect in regenerative processes including cellular proliferation, angiogenesis, fibrosis, and inflammation	[101]
Particle	Bone	AuNC23, macrophage membrane	Resolvin D1 AuNC increase M2 macrophage polarization	[106]
		Folic acid, silver	FA-AgNPs 24induce M1 macrophages reduction and M2 macrophage polarization	[108]
		PLGA, Resveratrol	Resveratrol released by 3D microsphere mediate M1 to M2 macrophage plasticity	[109]
		Europium,MSN	Europium-doped MSNs activate the osteogenic differentiation of MSC as well as angiogenic activity of HUVECs25	[111]
		Copper, MSN	Copper-doped MSNs activate OSM26 pathway	[112]
		MCP-1, MSN, FasL	TDNs27 enables rapid release of MCP-1 to recruit activated T cells and then induces their apoptosis through the conjugated FasL28	[113]
	Heart	PLGA, Pitavastatin	Enhanced vascular permeability, Activation of Anti-Inflammation	[107, 110]

¹MCPC: Magnesium–calcium phosphate cement

²TGF-β1: Transforming growth factor beta-1

³IL-6: Interleukin-6

⁴IFN-γ: Interferon gamma

⁵SrBG: Strontium bioactive glass

⁶BMP-4: Bone morphogenetic protein-4

⁷MSNs: Mesoporous silica nanoparticles

⁸Aker/SA: Akermanite/Sodium alginate

⁹Alg/Ser/GO: Alginate/Sericin/Graphene oxide

¹⁰GelMA: Gelatin methacrylate acid hydrogel

¹¹PRP: Platelet-rich plasma

¹²GAG: Glycosaminoglycan

¹³PEG: Poly(ethylene glycol)

¹⁴MCP-1: Monocyte chemotactic protein-1

¹⁵IL-8: Interleukin-8

¹⁶MIP-1α: Macrophage inflammatory protein 1 alpha

¹⁷MIP-1β: Macrophage inflammatory protein 1 beta

¹⁸dECM: Decellularized extracellular matrix

¹⁹xNDM: Xenogeneic native porcine brain decellularized extracellular matrix

²⁰PLGA: Poly(lactic-co-glycolic acid)

²¹PME: Porous pneumatic microextrusion

²²PDRN: Polydeoxyribonucleotide

²³AuNC: Gold (Au) nanocage

²⁴FA-AgNPs: Folic acid-silver (Ag) nanoparticles

²⁵HUVECs: Human umbilical vein endothelial cells

²⁶OSM: Oncostation M

²⁷TDNs: T-cell depleting MSNs

²⁸FasL: Fas ligand

Macroscopic 3D scaffolds

Biocompatible materials have been used to generate rigid 3D scaffolds that provide biochemical and physical support for tissue regeneration. For long-term stable retention and replacement with regenerated tissue, biodegradable and biocompatible polymers such as poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), and polycaprolactone (PCL) have been widely used in the manufacture of diverse scaffolds for treating various defects. In addition, inorganic materials and minerals also have been regarded as excellent candidates for 3D scaffold fabrication, especially for bone tissue regeneration owing to their osteoconductivity and biocompatibility [70]. Calcium phosphate is a representative mineral containing inorganic phosphate anions, demonstrating various bioactivities [71]. Derivatives of calcium phosphate, including magnesium–calcium phosphate (MCP) and biphasic calcium phosphate (BCP), were manipulated into scaffolds and provided a favorable immune microenvironment for bone tissue regeneration [72, 73]. The MCP scaffold inhibited the expression of inflammatory cytokines including TNF-α and IL-6, while increasing the production of pro-regenerative cytokines such as bone morphogenic protein (BMP)-2 and TGF-β. The consequent interaction between the scaffold and macrophages induced osteogenesis of stem cells and angiogenesis, which are crucial processes for new bone formation [72]. In addition, BCP scaffolds can regulate innate and adaptive immune responses to promote new bone formation by directing the polarization of macrophages into the M2 phenotype and inhibiting the maturation of dendritic cells [73]. Moreover, BCP scaffolds incorporating supplementary nanomaterials that enable controlled release of dual immunomodulatory molecules (IL-4; M2 macrophage polarization inducer, dexamethasone; dendritic cell maturation inhibitor) provide a desirable microenvironment for optimal immune responses and consequent osteogenesis [73].

Another study reported the importance of sequentially polarizing macrophages from M1 to M2 during bone regeneration by developing a PLGA-based composite scaffold containing interferon-γ and strontium-doped bioglass, which induces M1 polarization and M2 activation, respectively [74]. The controlled biochemical microenvironment successfully mediated the transition of the macrophage phenotype, and the sequential activation of macrophages further enhanced osteogenic differentiation and new bone formation [74].

The development of functional scaffolds using rigid bioactive materials has been accelerated by cutting-edge fabrication techniques such as 3D printing and micro-molding. The infinite tunability of the structure and function of these scaffold systems would further improve the efficacy of immune regulation in a precisely controlled manner for versatile tissue regeneration.

Hydrogels

Hydrogel, a water-containing polymeric network, is one of the most popular biomaterial systems used in various biomedical applications. In particular, hydrogel-based therapeutics are advantageous in terms of the fabrication of 3D constructs with various shapes and non-invasive in vivo applications. Moreover, adjustment of crosslinking method can vary the way of applications and physicochemical properties of each hydrogel, which allow facile tunability depending on final targets. Tissue-like hydrogel systems in tissue remodeling could significantly improve cellular engraftment and maintenance of transplanted cells at the wound site for enhanced therapeutic results. In general, different tissues have different microenvironments that play crucial roles in cellular differentiation and activities and thereby, maintain homeostasis in the tissues. In addition to the biochemical composition (e.g., ECMs and cytokines), tissue-specific mechanical properties also significantly affect the phenotypic changes and functionalities of cells. Recently, it was revealed that mechanical cues could induce the polarization of macrophages and alter their behaviors [75–77]. When macrophages were cultured on polyacrylamide gels with various stiffnesses, the stiffer substrates induced macrophage polarization into a pro-inflammatory phenotype [76]. Another study also reported significant upregulation of pro-inflammatory activation and related cytokine secretion through integrin-mediated signaling under increased stiffness by ECM crosslinking [77].

As biochemical cues, various natural or synthetic polymer-based hydrogel systems have been used to regulate immune reactions and the remodeling of various tissues. Alginate is a natural polymer that has been widely used in various biomedical applications owing to its biocompatibility and straightforward crosslinking mechanism [78]. By combining with bioactive molecules, alginate hydrogel has demonstrated potential in immunomodulation for successful skin regeneration [79, 80]. Alginate hydrogels incorporating bioactive glass have been demonstrated to polarize macrophages into the M2 phenotype and subsequently improve ECM synthesis and vascularization, which mediates successful skin wound regeneration with minimized scar tissue formation [79]. Ackermanite-containing alginate hydrogel also induced the activation of anti-inflammatory macrophages and recruited stem cells extensively (which is a crucial process for tissue remodeling) [80]. In other studies, ECM molecules such as gelatin and heparin were used to fabricate hydrogel systems for skin wound treatment. Liang et al. reported that ultraviolet (UV)-crosslinkable silk fibroin hydrogel showed higher regenerative capacity in skin wounds than alginate-based hydrogel systems with minimized scars by recruiting pro-regenerative macrophages [81]. In addition, hydrogel combined with poly(ethylene glycol) and heparin (a highly anionic glycosaminoglycan) could absorb anti-inflammatory cytokines for immune cell invasion when applied as a wound dressing on a skin wound. The trapped cytokines then, enhanced vascularization and wound healing [82].

For bone regeneration, hydrogel as well as supplements for improved mechanical and osteoconductive properties have been implemented to maximize therapeutic results. Jiang et al. prepared an injectable hydrogel system composed of alginate, sericin, and graphene oxide (GO) for bone defect applications [83]. Sericin, a glycoprotein produced by silkworms, is known to reduce inflammation and host foreign body reactions. In addition, GO, oxidized graphene has been utilized extensively for tissue regeneration and disease treatment based on its biocompatibility, anti-oxidation, protein absorption, and osteoconductive properties [84, 85]. The synergistic role of sericin and GO in alginate hydrogel substantially improved the migration and activation of M2 macrophages, and the proliferation, osteogenesis, and mineralization of MSCs, which resulted in enhanced new bone formation) [83]. Gelatin, a collagen-derived ECM-like polymer, has also been modified and widely used in hydrogel-based bone defect regeneration approaches. As a hybrid format of gelatin hydrogel, various bioactive molecules including bone morphogenic protein-4 (BMP-4), silica nanoparticles, nanosilver, halloysite, and nano fish bone were included to improve the mechanical properties and osteoconductivity of the hydrogel system. More importantly, the reduced pro-inflammatory responses and increased regenerative immune reactions with M2 polarization were accomplished by incorporating bioactive molecules, inducing successful bone defect reconstruction [86–89]. In addition, gelatin hydrogel-based immunotherapy has also shown potential in cartilage regeneration. The methacrylated gelatin hydrogel was supplemented with platelet-rich plasma (PRP) and investigated as a bio-ink for 3D printing [90]. PRP-incorporating gelatin hydrogel could orchestrate the induction of anti-inflammatory immune responses and subsequent tissue remodeling through enhanced stem cell proliferation, migration, and cartilage formation [90].

Although it would be difficult to directly determine which base material is the best for regenerative immunomodulation, previous studies have indicated that various hydrogels could be developed into an effective platform for regulating the immune microenvironment and remodeling in various tissues. Many of the current hydrogel systems for regenerative immunomodulation are aimed at inducing anti-inflammatory responses by exploiting M2 macrophage polarization and secreted cytokines. However, because the immunity during the healing process is dynamic and complex, the relationship between the materials used and their roles in regeneration should be clarified further. In addition, as one of the advantages of hydrogels, precise control of the properties of the system would improve the therapeutic efficacy of immune regulation for successful tissue regeneration.

Decellularized tissues

Decellularized extracellular matrix (dECM) has been highlighted in tissue engineering and regenerative medicine because it can mimic a complex and elaborate tissue microenvironment that cannot be fabricated conveniently using conventional synthetic approaches [91]. In addition, significantly high biocompatibility and minimized concern of immune rejection have further broadened the application of dECM in the fabrication of versatile formats of 3D scaffold systems [91–96]. Recent studies have reported that cell culture substrates composed of lymph node extracellular matrix (LNEM) could recapitulate biochemical microenvironment of lymphatic tissue and increase M2 macrophage polarization compared with collagen-based culture substrates [93]. The immunomodulation effect was enhanced further in the 3D LNEM hydrogel system. In addition, the macrophages in the construct demonstrated significantly increased viability, phagocytosis, M2-type marker expression, and anti-inflammatory cytokine secretion compared with collagen or ECM hydrogel system derived from other tissues. Finally, the immunomodulatory properties of the LNEM hydrogel improved M2 macrophage recruitment and muscle tissue regeneration in a defect when these were applied to a mouse model of volumetric muscle loss [93]. When combined with stem cell therapy, dECM scaffolds further enhanced the induction of anti-inflammatory responses and reduction of pro-inflammation, resulting in histological and functional restoration of muscle damage [97].

Based on tissue-specific biochemical composition, tissue-derived dECM has been utilized in tissue-specific applications. dECM from porcine brain tissue promotes the restoration of behavioral activity and reduces the size of lesions of spinal cord injury [98]. Similarly, cartilage-derived dECM scaffold improved the participation and chondrogenesis of stem cells in knee osteochondral defects [99]. In addition to the regenerative property of dECM materials, both the studies verified a significant increase in the population of M2 macrophages and anti-inflammatory cytokines such as IL-10, arginase-1, and TGF-β, demonstrating controlled immune microenvironment of defects into pro-regenerative conditions by using dECMs.

Moreover, the incorporation of anti-inflammatory molecules such as IL-4 [99] and rosiglitazone [100] in dECM-based scaffolds could further improve immunomodulatory effects and efficacy of regeneration in cartilage and ligament, respectively. dECM can also be integrated into a polymeric scaffold for renal regeneration. Decellularized kidney matrix was introduced into a PLGA scaffold and implanted at a defect after nephrectomy [101]. Although this study reported the synergistic role of dECM in regulating inflammation and regeneration of renal tissue with other bioactive molecules including magnesium hydroxide, polydeoxyribonucleotide, and cell-derived extracellular vesicles, the integrated bioactive scaffold made of dECMs could provide an optimal microenvironment for pro-regenerative immune reactions [101]. Combining previous studies, in addition to the strong regenerative and tissue-specific properties, induction of anti-inflammatory responses by dCEM could be implemented in bioactive scaffolds to empower regenerative immunotherapy.

Particles

Micro or nanosized particle systems have been implemented extensively in various biomedical applications including biosensors [102, 103], cell carriers [104, 105], and drug-eluting systems [106, 107]. Particle-based therapeutics are particularly advantageous in non-invasive in vivo applications. Moreover, modification of the biochemical properties of particles is relatively convenient, thereby providing specific functionalities for specific applications. Drug-containing nanoparticles composed of Au or Ag have been developed into immune modulators, which efficiently induce M2 macrophage polarization and the remodeling of bone and cartilage tissues [106, 108]. Specifically, gold nanocage was coated by cell membrane of macrophages with cytokine receptor, and the surface thereby circumvent pro-inflammation activation [106]. The controlled release of Resolvin D1, a M2 polarization inducer, from the engineered gold nanocage could efficiently develop reparative immune microenvironment and induce new bone formation [106].

PLGA has been a popular material for particle fabrication because of its great biocompatibility and versatility in sustained drug release. Therefore, it has been applied in the control of regenerative immunity by incorporating compounds for immunomodulation. Resveratrol is a natural compound that is known to induce M2 macrophage polarization and osteogenesis [109]. Resveratrol-containing PLGA microspheres continuously released molecules to convert pro-inflammatory macrophages to the M2 phenotype (secreting cytokines related to anti-inflammation and neovascularization) and thereby, induced osteogenic differentiation [109]. After combining the microparticles with a 3D scaffold, the study suggested the combinatorial scaffold system as an immune engineering platform for bone tissue regeneration [109]. Other studies reported PLGA nanoparticles encapsulating pitavastatin (pitavastatin-NP) for treating acute myocardial infarction (MI) [107, 110]. When Pitavastatin-NP targeting monocytes were injected serially, the accumulation of monocytes and macrophages in the defects decreased significantly, and in turn, resulted in enhanced left ventricular remodeling. The phenomenon indicates the availability of a clinically feasible strategy for post-MI treatment [107, 110].

Mesoporous silica nanospheres (MSNs) have been used as a popular nanomaterial for drug-eluting systems. In particular, various derivatives of MSNs with chemical modifications have been developed and exploited for immunomodulation for tissue regeneration [111, 112]. Through a facile one-pot method, bioactive ions such as copper and europium were doped onto MSNs and treated for bone tissue remodeling [111, 112]. As a result, the particles upregulated inflammatory responses of macrophages and angiogenesis and thereby, induced osteogenesis mainly through activation of the oncostatin M (OSM) signaling pathway [112]. It is noteworthy that unlike previous studies, which mostly focused on the activation of anti-inflammatory reactions, these studies targeted the induction of pro-inflammatory reactions for OSM signaling and subsequent osteogenesis [112]. Although long-term toxicity and a comprehensive understanding of the biological function of the ions used in immunomodulation should be evaluated further, the ion-doped MSNs suggested a facile and effective immune therapy for bone remodeling [111, 112]. To treat bone loss caused by chronic inflammation, another study modified the surface of monocyte chemotactic protein-1 (MCP-1)-containing MSNs with Fas-ligand that can specifically target activated T-cells [113]. After introducing the particles, MCP-1 was released from the particle and induced T-cell apoptosis and M2 macrophage transformation [113]. The regulation of the balance between Treg and Th17 cells, which play important roles in the homeostasis of bone tissue, could demonstrate the potential of MSNs in treating immune disorders [113]. As a therapeutic platform, many researchers have attempted to identify optimal core materials and adjust the physicochemical properties of particles with functional moieties to efficiently control the immune microenvironment.

Future direction of biomaterial-based regenerative immunotherapies

Here, we reviewed the utilization of functional biomaterials in modulating the immune microenvironment that facilitates tissue regeneration. Due to the potential of countless and spatiotemporal designs for various applications, the use of biomaterials could greatly enhance the efficiency and efficacy of regenerative immune therapies. As a powerful tool to precisely control mechanical, topological, and biochemical cues, biomaterial-based platforms also have extended our understanding on the underlying mechanisms of immunomodulation for successful tissue remodeling. However, a large portion of the correlation between the used materials and the complex immune system still remains unknown. Therefore, approaches simply increasing anti-inflammatory responses, which is one of the major targets of current biomaterial-based immune therapies, could limit the therapeutic potential and trigger unexpected side effects. In addition, long-term safety, and off-target effects of the used biomaterials, especially in the systemic administration of particle-based systems, should be comprehensively investigated for successful translation into clinical applications.

Conclusion

In summary, we investigated recent engineering approaches applied in the modulation of regenerative immunity. Direct and indirect regulation of the immune microenvironment by various cell types and biomaterials have shown significant advances and opened a new strategy in the field of tissue regeneration and disease treatment. The increased population of regenerative immune cells and secreted cytokines after the treatments can reduce inflammation at the lesion and support tissue remodeling through the induction of vascularization and ECM reorganization. Based on the accumulated knowledge on the dynamic crosstalk between the immune system and immunomodulatory treatments, various cutting-edge engineering tools would be deployed in designing optimal regenerative immune therapies. Moreover, the advanced techniques precisely controlling the duration of immune response would maximize the efficacy of regenerative engineering techniques and thereby, potentially translate these into clinical applications.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Ethical statement

There were no animal or human subject experiments carried out for this article.