Unraveling the potential of 3D bioprinted immunomodulatory materials for regulating macrophage polarization: State-of-the-art in bone and associated tissue regeneration

Abstract

Macrophage-assisted immunomodulation is an alternative strategy in tissue engineering, wherein the interplay between pro-inflammatory and anti-inflammatory macrophage cells and body cells determines the fate of healing or inflammation. Although several reports have demonstrated that tissue regeneration depends on spatial and temporal regulation of the biophysical or biochemical microenvironment of the biomaterial, the underlying molecular mechanism behind immunomodulation is still under consideration for developing immunomodulatory scaffolds. Currently, most fabricated immunomodulatory platforms reported in the literature show regenerative capabilities of a particular tissue, for example, endogenous tissue (e.g., bone, muscle, heart, kidney, and lungs) or exogenous tissue (e.g., skin and eye). In this review, we briefly introduced the necessity of the 3D immunomodulatory scaffolds and nanomaterials, focusing on material properties and their interaction with macrophages for general readers. This review also provides a comprehensive summary of macrophage origin and taxonomy, their diverse functions, and various signal transduction pathways during biomaterial-macrophage interaction, which is particularly helpful for material scientists and clinicians for developing next-generation immunomodulatory scaffolds. From a clinical standpoint, we briefly discussed the role of 3D biomaterial scaffolds and/or nanomaterial composites for macrophage-assisted tissue engineering with a special focus on bone and associated tissues. Finally, a summary with expert opinion is presented to address the challenges and future necessity of 3D bioprinted immunomodulatory materials for tissue engineering.

Graphical abstract

Highlights

• •Effects of 3D immunomodulatory platforms for tissue healing and regeneration via regulating macrophage fate.
• •Biomaterial-mediated targeted immune response via multiple signaling pathways.
• •The role 3D bioprinting in macrophage-assisted bone and associated tissue regeneration.
• •Next-generation smart immunomodulatory platforms for sustained delivery of biomolecules for precision immunoengineering.

1. Introduction

The human immune system plays an essential role in tissue regeneration and disease progression. Macrophages were the first phagocytes discovered by Mechnikov and Herlich in 1908 (Nobel Prize in Physiology or Medicine) [1,2]. Macrophages are associated with the host immune response through various internal or external stimuli and act as effectors for other immune cells. Macrophages are also crucial mediators of various physiological and pathological conditions, such as inflammation, acute infection, and tumors [3]. In recent years, significant efforts have been devoted to the fabrication of various immunomodulatory biomaterials for tissue regeneration. Immunomodulatory biomaterials not only interact with macrophage cells but also elicit host-specific immunity (implant-mediated immune response) and regulating the fate of macrophages [4]. The surface topology (stiff or soft matrix) [[5], [6], [7], [8], [9]], chemical composition [[10], [11], [12]], particle size [10,13,14], porosity [15,16], self-assembly [17,18], wettability [[19], [20], [21], [22]], and roughness [23,24] of the biomaterial promote the specific immune response. Moreover, the degradation products of biomaterials may exhibit various immunomodulatory effects on immune cells, which can initiate a local immune response at the implantation site [4]. After the implantation of a biomaterial scaffold, innate immunocytes (undifferentiated monocytes or M0 macrophages) arrive at the scaffold surface. Based on the scaffold wettability, various proteins, such as collagen, fibronectin, fibrinogen, and vitronectin, accumulate on the scaffold surface and initiate the blood coagulation process through a series of signaling pathways. The characteristics of implanted biomaterials may influence the onset, intensity, and outcome of acute or chronic inflammatory responses [25]. For more information about the inflammatory response of biomedical implants, a reader is encouraged to study the most cited paper, ‘Inflammatory response to implants’ [26]. In the event of unrestrained or prolonged inflammation or the absence of bioactive cues, foreign body reaction/response (FBR) can result in fibrous encapsulation of implants, a sequence involving monocyte recruitment, macrophage activation, polarization, and integration into giant cells, to separate them from their surroundings and inhibit their direct interactions. Adaptable/suitable material properties, mechanical characteristics, physical cues, chemical functions, and biological activities play a crucial role in providing regulatory signals to guide the destiny of macrophages in response to biopolymers [[27], [28], [29]]. However, integrating all physical and chemical properties into a single platform for boosting immunity remains challenging owing to the low availability of biocompatible polymers. Duan et al. showed that prolonged exposure of titania nanoparticles may decrease the activity of T-lymphocytes (CD3⁺, CD4⁺, and CD8⁺), NK cells, B-lymphocytes, and decreased the level of IL-2 in serum, indicating the anti-inflammatory activity [30]. Therefore, an ideal biomedical implant must have high immunomodulatory efficacy with extremely low site-specific or systemic toxicity in vivo.

Besides scaffold chemistry, micro/nano-fabricated scaffolds have gained significant attention for developing immunomodulatory platforms. One of the exciting biofabrication strategy is ‘Additive manufacturing (AM)’ or ‘three-dimensional (3D) printing’ or ‘rapid prototyping’. 3D printing combines versatile materials (e.g., polymers, ceramics, powders, metals, and composites) into a single platform to create various biomimetic structures for industrial applications [[31], [32], [33]]. 3D bioprinting can be applied to tissue engineering and regenerative medicine to address the need for biomimicking the structures of native tissues by cell-laden culture and allowing us to study immunomodulation dynamically. However, the major question arise in 3D printing/bioprinting is – ‘are the 3D structures safe for implantation or capable of generating host-specific immunity?’ Various 3D immunomodulatory platforms have been reported for soft (e.g., skin, muscle, heart, lung, and meniscus) and complex (e.g., bone and cartilage) tissue engineering. Metal/ceramic-based composites are only suitable for bone tissue regeneration and cannot be used for soft tissue engineering. Furthermore, most commercial or clinically approved biomedical implants are titania or hydroxyapatite (HAp)-based in the case of bone, and alginate, collagen, or decellularized extracellular matrix (dECM)-based in the case of soft tissues [34,35]. Importantly, the existing 3D scaffolds or bioprinted hydrogels fail to demonstrate the immune-triggering effect due to poor biodegradability, a higher chance of foreign body response (FBR), and inflammatory cells production in vivo [36].

To our knowledge, most 3D micro/nano-fabricated scaffolds that exhibited excellent tissue regenerative capabilities are ill-explored towards in vitro and in vivo immune response. The existing literature mainly reported using various micro/nano-fabricated scaffolds with immunomodulatory properties via culturing either mouse or human cells without proper demonstration of in vivo immunomodulation. Furthermore, the underlying signaling pathway is ill-explored and limited to titania-based implants. Anderson et al. previously demonstrated an insightful review in 2008 regarding the use of synthetic biomaterials and their effect on macrophage-mediated immune response [29]. This work overviews new concepts and understandings regarding host immune response to various biomaterials.

Similarly, Li et al. [37] recently demonstrated the biomaterial design strategies, mainly the biomaterial properties on macrophage polarization, adhesion, migration, and tissue regeneration. This review provides an overview of only biomaterial properties but not focusing on any particular fabrication technology-based macrophage polarization strategies. Moreover, Whitaker et al. also reported using various immunomodulatory biomaterials for tissue repair with a particular focus on wound healing only. None of the previously published articles illustrated macrophage immunomodulation's underlying molecular mechanism (signaling pathways involved in macrophage polarization) during complex tissue regeneration. This review summarized the use of various implantable biomaterials and their physicochemical properties for macrophage polarization. Fig. 1 briefly illustrates an overview of the design strategy of different immunomodulatory biomaterials and their impact on macrophage immunomodulation. Not only that, this review also focuses on demonstrating the signaling pathways associated with biomaterial-macrophage interaction for understanding both material scientists and clinicians for developing next-generation innovative and stimuli-assisted immunomodulatory platforms. Lastly, this review illustrates the recent progress in 3D printed immunomodulatory scaffolds for tissue engineering, focusing on bone regeneration, followed by an insightful summary and future direction.

Fig. 1.

Schematic illustration of the immunomodulation strategies for tissue engineering. The interaction of immune cells (e.g., macrophages) and their dynamic polarization in respond to micro/nano biomaterials. Future research must focus on the development of novel immunomodulatory biomaterials with tissue specificity and integrating advanced biofabrication tools (e.g., 3D printing and bioprinting) and genome editing tools (e.g., CRISPR/Cas9) for precision immunoengineering.

2. Macrophage heterogeneity - tissue healing or disease progression?

2.1. Origin of macrophages – tissue residents and immigrants

Classically, macrophages are specialized phagocytes mainly involved in wound repair and regeneration. Macrophages travel through the bloodstream for immune surveillance and can be divided based on tissue specificity: skin (skin-resident macrophages, Langerhans cells), bone (bone marrow macrophages and dendritic cells), heart (cardiac macrophages), lung (alveolar macrophages), kidney (medullary macrophages), liver (Kupffer cells), intestine (intestinal macrophages), lymph (lymphatic macrophages), peritoneum (peritoneal macrophages), brain (glial macrophages), and eye (intraocular macrophages) [38]. These tissue-resident macrophages are originally derived from the ectoderm or fetal liver. Macrophages originate from monocytes derived from the adult and neonatal bone marrow. During late embryogenesis, macrophages colonize various tissues. Owing to the remarkable self-renewal (self-proliferation and differentiation capability) properties of macrophages, they are mostly retained in adult tissues [[39], [40], [41]]. The microglial cells of the central nervous system (CNS) and Langerhans cells of the epidermis are recognized as true ‘tissue resident macrophages’ (Fig. 2a). However, there is a debate about if and to what extent the true tissue-resident macrophages are still present in adult tissues. In mice, around 2–4% of the total circulating leukocytes are recruited in most tissues to replenish the resident macrophages during healthy conditions or acute inflammation. Murine monocytes are differentiated from specialized cells called lymphocyte antigen 6 (LyC). During normal conditions, the LyC⁻ monocytes from bone marrow, liver, and spleen travel in the blood stream for immune surveillance. During pathogen invasion, the LyC⁻ cells differentiated into LyC⁺ monocytes and induced the secretion of CCL2 chemokines (Fig. 2b). Without inflammation or pathogens, the LyC- cells replenish in gut, liver, spleen, skin, and lung or return to bone marrow or die after 2–3 days.

Fig. 2.

Origin and classification of macrophages. (a) Monocytes derived from embryonic tissue differentiated into microglial cells (CNS resident cells) and Langerhans cells and later transformation into CNS resident macrophages. The resident macrophages later polarized into choroid plexus, leptomeningeal, and perivascular macrophages. (b) Macrophages are also derived from Kupffer cells of the fetal liver and subsequently differentiated into M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages and have self-renewal properties. (c) Macrophage polarization and its various sub-types. Classically activated macrophages (M1) are involved in pathogen killing, foreign body reaction, Th1 effector production, and tumor inhibition. Alternatively, activated macrophages (M2a, M2b, and M2c) are involved in immunomodulation, osteogenesis, matrix deposition, Th2 effector production, and tumor progression.

2.2. Macrophage polarization

Macrophage phenotypic plasticity is profoundly affected by stimuli from the internal or external environment. Macrophages switch to various phases during tissue healing and regeneration, thus considered primary therapeutic targets for multiple diseases [38]. During inflammation, macrophages migrate to wounded tissue and differentiate (polarize) to repair inflammation [42]. Macrophages are divided into two categories based on their nature and function: M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages (Fig. 1c). M1 macrophages are usually activated through various pro-inflammatory signals, such as tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), and lipopolysaccharide (LPS), leading to the secretion of various pro-inflammatory cytokines. Proinflammatory cytokines and chemokines trigger immunity against pathogenic attacks or malignancies by augmenting reactive oxygen species (ROS) and nitric oxide (NO). Natural killer (NK) and helper T-1 (Th1) cells regulate the phenotypic plasticity of M1 macrophages through TNF-α secretion [[43], [44], [45], [46]]. NK cells transiently secrete TNF-α, whereas Th1 cells secrete TNF-α sustainably during a steady-state immune response against microbial pathogens. The remarkable immunity-boosting efficacy of M1 macrophages makes designing various therapeutic platforms easy [38]. For example, Gill et al. used genetically modified M1 macrophages with chimeric antigen receptors (CAR) to enhance the phagocytic function against cancer cells [47]. CAR macrophages express higher levels of pro-inflammatory cytokines and chemokines than primary dendritic macrophages. Furthermore, in addition to killing tumor cells, CAR macrophages can sense and kill healthy tissues. Moreover, many therapeutic biomaterials may trigger local inflammation upon transplantation; therefore, balancing CAR-macrophage and primary macrophages in the human body is crucial for regulating the immune response [47]. Anti-inflammatory macrophages (M2 macrophages), on the other hand, are activated by Th2 cytokines, such as IL-4 and IL-13 (secreted mainly by mast cells, T-lymphocytes, eosinophils, basophils, and neutrophils), which is entirely different from the Th1 activation axis [[48], [49], [50], [51]]. M2 macrophages are primarily associated with wound healing (epithelial macrophages) and tissue regeneration (regulatory macrophages). M2 macrophages have various subtypes: M2a, M2b, and M2c [52]. Table 1 and Table 2 depict an overview of various macrophage phenotypes in vitro and in vivo systems with their activation modes and functions in tissue regeneration and therapy.

Table 1.

A summary of various in vitro macrophage phenotypes, their activation modes, and major secretory products reported in the literature [37,45,48,[53], [54], [55], [56], [57]].

Phenotypes	Modulator/Inducer	Surface markers	Intracellular markers	Cytokines	Chemokines
M1a	TNF-α	CD86High	iNOSHigh PTGS2High	IL-6high	CCL8high
	LPS	MHC-IIHigh		IL-1high	CCL15moderate CCL20high
	IFN-γ	IL-2RaHigh		IL-10low	CXCL9high
		IL-7RHigh		IL-12high	CXCL10Low
				IL-15moderate	CXCL13Low
				TNF-αhigh
M2ab	IL-4	MHC-IIHigh DCL-1High	Arg-1High PTGS1High	IL-10moderate	CCL13High
	IL-13			IGF-1high	CCL14High
				TGF-βhigh	CCL17Moderate
				FN1moderate	CCL18High
				IL-1rahigh	CCL23High
				IL-6low	CCL26High
M2bc	IL-1β	MHC-IIHigh CD86Low	SPHK1High	IL-1low	CCL1Moderate
	TLR			IL-4moderate	CCL20High
	LPS			IL-6low	CXCL1High
	IL-1R			IL-10high	CXCL2Moderate
				IL-12low	CXCL3Low
				TNF-αlow
M2cd	IL-10	CD163High TLR-1Low	SLAMHigh	IL-10high	CCL18High
	TGF-β	TLR-8High CD206High	MRHigh	TGF-βmoderate	PTX3Low
	various glucocorticoids

^aTh1 activation, pro-inflammatory function (Type-I immune response), phagocytic, pathogenesis, and tumor clearance.

^bTh2 activation, anti-inflammatory (Type-II immune response), anti-parasitic, host-specific immunity.

^cTh2 activation, anti-inflammatory (Type-III immune response), help in antibody production via immune cross-talk with B lymphocytes), tissue healing through immunomodulation.

^dTh2 activation, anti-inflammatory (Type-III immune response), tissue healing and regeneration, matrix mineralization, inhibition of acute inflammation.

Table 2.

Summary of various in vivo macrophage phenotypes and their identification markers reported in the literature [58].

Phenotypes	Effector molecule	Identification markers
Primary monocytes	F8/80+, MHC-II+, CSF-1R+	CD11b+, CD115+
Inflammatory/circulating monocytes	CX3CR1Low, CD62L−, CD43+, Cd11c-	Ly6C+, CCR2High
FBRa macrophages	CD64Moderate, MHC-II+	CD6High, F4/80High
M1 macrophages	pSTAT1, pSTAT5, iNOSHigh, IL-1R1, TLR2, TLR4, IRF-5, IL-12High, IL-23High, TNF-αHigh, IL-1βHigh, IL-6Moderate, CCL(2,3,4,5,8,9,10,11), CXCL9, CXCL10, CXCL11	CD80+, CD86+, CCR7High, SOCS3
M2a macrophages	CD200R?, Stabilin-1, CD301, Dectin-1, SR, IL-1R, Polyamine, CCL17, CCL22, CCL24, TGF-βModerate	IL-10High, CD163High, SOCS1/2, Arg1High, CD206High, THM2, Fizz1Moderate, Ym1/2, pSTAT6
M2b macrophages	MHC-II, IL-1, IL-6, CCL1, TNF-α	CD86+
M2c macrophages	CD163, TGF-βHigh	MERTK, pSTAT3, CD206High
M2d macrophages	IL-10Moderate, IL-12, TNF-α, and TGF-β	VEGF-A
FBGCb	CD11c, CD44, CD98, CD206, E-cadherin, DC-STAMP, DC-SIGN, HLA-DR, B7-H1	CD86, MMP9
Anti-fibrotic macrophages	Arg-1-, CD206, FIZZ1, MERTK, CD74, CXCR2, CXCR4, MFGE8, MMP(3,8,9,12,13,14)	Arg-1+, CD280High, PPARγ

^aFBR, foreign body response.

^bFBGC, foreign body giant cell.

2.3. Macrophages polarization and soft tissue regeneration

Skin is one of the versatile tissue in our body. Skin wound healing is a complex biological process that comprises a series of signaling mechanisms and the activity of various skin cells. Previous research indicates that epidermal macrophages promote skin regeneration through the rapid proliferation of dermal fibroblasts and maximize protection against ROS. Macrophages eat up old or damaged cells through phagocytosis, killing the skin-tissue-embedded pathogens, and clearing the apoptotic tissues from the injury site [59,60]. M2-polarized macrophages secrete matrix metalloproteinases (MMPs) at the inflammation site [61]. Duffield et al. showed that the reduction of epidermal macrophages at the wound site delayed wound healing, leading to inefficient wound repair [62]. In contrast, M2 macrophages overexpressing the colony-stimulating factor (CSF), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor-α (VEGF-α) have been shown to promote robust wound healing via neo-angiogenesis and skin re-epithelialization. Moreover, adjacent parenchymal and stromal cells crosstalk with macrophages during wound healing. Furthermore, M2 macrophages promote the proliferation and differentiation of other stem cells and local progenitor cells, which indirectly regulates the macrophage phenotype [[63], [64], [65], [66], [67], [68], [69]] (Fig. 3a). In addition, M1 macrophages protect against pathogenic attack in the wound bed and are differentiated at the early stage of wound healing. However, hyperactivation of M1 macrophages may induce a fibrotic response and delay wound healing [70]. Therefore, a balance between the M1 and M2 phenotypes is critical for vascularized wound healing.

Fig. 3.

Schematic illustration of macrophage-assisted skin and bone regeneration. (a) The various stages of the inflammatory response during the wound healing process. M1 macrophages and T cells dominate the pro-inflammatory phase of wound healing, whereas M2 macrophages primarily govern the later stage. During this time, the M2 macrophages secrete various factors that induce the differentiation of fibroblasts, keratinocytes, and myofibroblasts [69]. (b) Macrophages are polarized through the stress or wound signal from the fractured area and polarized into the M1 phenotype (inflammatory phase) to inhibit pathogenic attack. During bone repair and remodeling, macrophages polarize into M2 phenotype and release anti-inflammatory cytokines, such as IL-10 and TGF-β, that stimulate the osteoblast/osteoclast differentiation via RANKL/OPG signaling axis. The M2 macrophage also promotes angiogenesis at later fracture healing stages [71].

2.4. Macrophages polarization and complex tissue regeneration

Among the hard tissues, bone is one of our body's most complex and largest tissues. The bone is a highly complex and dynamic tissue that harbors various hematopoietic and mesenchymal progenitor cells. Bone remodeling is a dynamic process involving bone resorption governed by osteoclasts—a later stage of osteoblasts. Osteoblasts are mainly involved in matrix mineralization (osteoid) and are later re-organized toward osteocytes, which act as mechano-transducers for controlling the calcium-phosphate turnover in the bone. Osteoid helps the mineralization process by incorporating calcium and phosphate, forming organic moieties, and producing characteristic hydroxyapatite crystals [[72], [73], [74]]. It is well known that bone cells are tightly connected with immune cells (bone marrow-derived dendritic cells, BMDCs), which play a significant role in osteoimmunity. Resident macrophages commonly associated with osteoclasts promote osteogenesis by inducing mineralization [75]. Bone marrow-derived macrophages (BMDMs) play a critical role in intramembranous and endochondral bone regeneration through the receptor activator of nuclear factor kappa-B/osteoprotegerin (RANKL/OPG) signaling axis. Mesenchymal stem cells (MSCs) secrete various cytokines and growth factors, such as VEGF, TGF-β, and IGF-1, which trigger resident macrophages to polarize toward the M1 or M2 axis. During the early stage of bone fracture healing, the neutrophils are recruited at the fracture site and induce the M1 polarization of macrophages (Fig. 3b). The M1 macrophages play a protective role in this phase against pathogenic attack [71]. In the late stage of bone regeneration, the M2-polarized macrophages induce the secretion of TGF-β and IL-10, thereby inducing angiogenesis and osteogenesis [[76], [77], [78], [79]]. Moreover, MSCs secrete IL-6, which triggers the sustained release of bone morphogenic protein-2 (BMP-2) and promotes the phosphorylation of suppressor of mothers against decapentaplegic (SMAD) via the TGF-β or MAPK signaling pathway in osteoblasts [80]. Therefore, the interplay between MSC immunomodulation and macrophage polarization regulates osteoblast maturation and bone mineralization.

3. Effect of biomaterial properties on macrophage polarization

3.1. Effect of biophysical properties on macrophage fate

3.1.1. Surface stiffness

Mechanical properties, such as surface stiffness and viscoelasticity are important parameters for the primary immune response [[81], [82], [83], [84], [85], [86]]. The stiffness of human tissues varies with age and the nature of the tissue at the time of healing, fracture, or disease. In this context, hydrogels with mechanically stiff matrices (∼600–850 kPa) have been shown to promote both pro-inflammatory and anti-inflammatory activation of macrophages, while the soft matrix (∼120 kPa; low stiffness) promotes only pro-inflammatory activation of macrophages in vivo [87]. Gloffin et al. reported that the elastic modulus of rat wound tissue increased from 18.5 kPa to 50 kPa after 12 days of wound closure through the activation of M2 polarized macrophages [88]. This study also highlighted that focal adhesion kinase-mediated α-smooth muscle actin (α-SMA) orientation in dermal macrophages is a key regulator of M2 polarization and subsequent wound healing. Moreover, activation of cell migration, gene expression, protein secretion, higher glucose metabolism, and cytokine secretion are higher in the presence of a stiff matrix than in the presence of a soft matrix in T cells [84]. The activation of mechanoreceptors (e.g., YAP, TAZ, RAC, and Rho GTPases) plays a pivotal role in macrophage immunopolarization [[89], [90], [91], [92], [93], [94]]. Therefore, by controlling biomaterial stiffness, macrophage fate can be easily manipulated toward tissue regeneration or disease progression.

3.1.2. Surface charge

Electrophoretic light scattering analysis revealed that the human cell membrane is negatively charged, with a potential ranging from −10 to −90 mV [95]. Positively charged biomaterials significantly affect the adhesion and proliferation of macrophages onto the surface and, therefore, affect protein adsorption and release. Generally, positively charged biomaterials (cationic) elicit anti-inflammatory responses (M2 polarization) in macrophages compared with negatively charged biomaterials (anionic). Negatively charged biomaterials usually trigger both pro-inflammatory and anti-inflammatory responses, and a switch from M2 to M1 occurs in the presence of a negatively charged surface [[96], [97], [98]]. Cationic scaffolds made from dextran, polylysine, polyethyleneimine (PEI), and gelatin trigger M1 polarization of RAW 264.7 cells via TLR-4 signaling and induce the secretion of IL-12, thereby inhibiting the growth of M2-like tumor-associated macrophages (TAMs) toward antitumor immunity [99,100]. Ding et al. reported that incorporating strontium-doped HAp (Sr-HAp) into a dextran/chitosan hydrogel promotes rapid bone regeneration through M2 macrophage immunomodulation owing to the presence of –NH₂ groups of chitosan or by controlled release of Sr²⁺ ions from the chitosan scaffold [101]. This study also indicated that a combination of Sr and HAp is mainly responsible for the phenotypic switching of RAW 264.7 cells in vitro. Similarly, Fan et al. reported that toosendanin (bark extract of Melia toosendan)-loaded dextran sulfate discs inhibit colitis infection via M2 macrophage polarization through activation of the NLR pyrine domain containing 3 (NLRP3) inflammasome and nuclear erythroid factor-2 (Nrf-2) signaling pathway [102]. 3D printed polylactic acid (PLA)/chitosan scaffolds with varying surface charges and geometries have also been shown to suppress the anti-inflammatory response in macrophages by secreting various pro-inflammatory cytokines, such as TNF-α and IL-6 [103]. The surface charge of nanocellulose can be tuned via grafting of various functional groups, such as carboxymethyl (anionic) or hydroxypropyltrimethyl ammonium (cationic) groups, to modulate the immune response [104]. Studies have shown that carboxymethyl cellulose (CMC) films induce monocytes to express the M1 phenotype, whereas hydroxypropyltrimethyl ammonium-modified films are unable to boost the inflammatory response to monocytes owing to their inert nature [105]. Cellulose nanocrystals (CNCs; anionic) have been shown to promote M1 polarization of dendritic macrophages through the activation of Th1-mediated signaling pathways [106]. Similarly, a study reported by Patel et al. showed that 3D printed CNC-modified chitosan/silk fibroin (SF) scaffolds promote osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) through M2 macrophage polarization via interacting with SF protein and chitosan [107]. This study also indicated that the net surface change of the composite scaffold tended to be positive and was probably the main reason for the inhibition of M1 polarization in RAW 264.7 cells. Moreover, charged nanoparticles (NPs) have a significant impact on macrophage polarization. The M2 macrophage polarization was due to the higher availability of the positive charges onto the scaffold surface (-NH₂ from chitosan and SF) and is independent to CNCs. Table 3 summarizes different NPs with varying surface charges and their role in macrophage polarization. Collectively, surface charge plays a significant role in macrophage phenotyping and could be a potential strategy to accelerate the immune response in the human body toward tissue healing and regeneration.

Table 3.

Effect of nanoparticle shape and surface charge on macrophage polarization for tissue engineering.

Type of nanoparticle	Size (nm)	Surface charge	Macrophage polarization	References
Silica NPs	10–1000	Negative	M1-type	[108]
Gold NPs	10–300	Negative	M1-type	[108]
Iron oxide NPs	30–280	Negative	M1-type	[108]
Polyurethane NPs	35–60	Negative	M1-type	[109]
NH2-polystyrene NPs	200	Positive	M2-type	[110]
PDA-modified Fe3O4 MNPs	107	Positive	M2-type	[111]
Hyaluronic acid-modified PEI-pDNA NPs	120	Positive	M2-type	[112]
Paramagnetic Fe3O4 MNPs	15–30	Positive	M2-type	[113]
Folate-modified Ag NPs	28	Positive	M2-type	[114]
Berbarin-loaded bilirubin-IgG NPs	15	Positive	M2-type	[115]
Poly-(lactic-co-glycolic) acid-Dox (PLGA-Dox) NPs	76	Negative	M1-type	[116]
Silymarin-PLGA NPs	180	Negative	M2-type	[117]
Fe3O4@C/MnO2 yolk-shell NPs	160	Negative	M2-type	[118]
Citrate-capped AuNPs	13–22	Negative	M2-type	[119]
Dox-loaded shMF NPs	150–160	Negative	M1-type	[120]

3.1.3. Chirality

Biological systems are composed of various chiral molecules, such as d-glucose, D/l-amino acids, L-phospholipids, and helical DNA, which play significant roles in the survival of an organism [121]. It has been shown that biomaterials integrated with chiral molecules facilitate various immune responses. Sun et al. demonstrated that chiral amino acids, such as N-isobutyryl-L(d)-cysteine-doped gold nanoclusters, promoted M1 polarization of dendritic macrophages and effectively inhibited the invasion of human proteolytic leukemia cells [122]. The modified chiral amino acid-coated gold nanoclusters exhibited higher macrophage adhesion efficiency, followed by filopodia and pseudopodia expansion and elicited the secretion of pro-inflammatory cytokines, thereby inhibiting the growth of fatal leukemia cells. In another study, Kehr et al. reported the use of D(l)-mannose-modified periodic mesoporous organosilica (PMO-D/L-MAN) nanoparticles and their effect on the in vitro polarization of human macrophages [123]. This finding also revealed that MAN-modified silica had 4-fold better adhesion and proliferation properties for macrophages than pure PMO. Furthermore, this study also demonstrated that the macrophage phenotype is not dependent on surface charge or functional group, but is dependent on mannose chirality, which helps in the adhesion and spreading of individual cells. Similarly, various chiral molecules have been shown to induce CD36 and TLR-2/6 markers in RAW 264.7 cells during differentiation. Collectively, the above-mentioned features demonstrate that surface chirality has a promising role in macrophage phenotyping and could be used as a potential target for the selective activation of immune cells.

3.1.4. Surface wettability

The physical properties of the biomaterial, such as wettability (hydrophilic or hydrophobic surface), may control the immune response of macrophages via regulating the adsorption of various proteins. Hydrophilic scaffolds facilitate the absorption of albumin, which can trigger the release of anti-inflammatory cytokines in M2 macrophages. In contrast, hydrophobic surfaces inhibit the absorption of proteins and therefore restrict macrophage activity [[124], [125], [126]]. For example, Zheng et al. reported that hydrophilic coating on the titania surface promoted the M2 polarization of RAW 264.7 cells by secreting various anti-inflammatory factors. This study further highlighted that hydrophilic coating on the titania surface facilitated the absorption and deposition of fibronectin, which triggered the phosphoinositide-3-kinase (PI3K) and NF-kB signaling pathways in RAW 264.7 cells, thereby conferring the M2 phenotype expression [9]. Titania nanotube (TNT) can be modified with superhydrophilic coatings through anodic oxidation and hydrogenation for selective activation of RAW 264.7 cells [127]. The hydrogenation of TNT leads to superior proliferation and adhesion of RAW 264.7 cells and facilitates the secretion of various anti-inflammatory factors, such as IL-10, BMP-2, and TGF-β. In another study, Webster et al. used a combination of hydrophobic and hydrophilic carbon nanofibers to modulate the pro-inflammatory response in primary T-cells [128]. Thus, biomaterial wettability plays a crucial role in the immune response, and manipulation of surface wettability may confer selective activation of immune cells toward tissue regeneration.

3.1.5. Surface topography

Surface topography is another important parameter that regulates macrophage fate [129]. Surface roughness (smooth or rough) and surface geometry (2D/3D: ordered/disordered or aligned/unaligned) play equally vital roles in cellular adhesion, proliferation, and differentiation. Compared to the patterned surface, the flat surface promotes a skewed macrophage morphology. Similarly, furrowed surfaces facilitate the elongation of cytoskeletal proteins of macrophages and alter cytokine expression [9,129]. It has been shown that grooved surfaces of polycaprolactone (PCL), polydimethylsiloxane (PDMS), and poly (lactic acid) (PLA), with average spacing of 250, 2,000, and 500 nm, respectively, force M0 macrophages to elongate and polarize into M2 macrophages. It has also been reported that microcontact printing of fibronectin onto the PDMS surface skews RAW 264.7 cells toward the M2 phenotype and enhances the expression of arginase-1 (Arg-1), CD206, and bronchoalveolar larval fluid protein (Ym1) [130]. Titania-based nanocomposites are frequently used in clinics as orthopedic or dental implants. The surface properties of titania-based implants can be tuned to manipulate the macrophage phenotype and enhance osteogenesis [131]. Refai et al. reported that sandblasted and acid-treated TNT promoted M2 polarization of RAW 264.7 cells and induced osteogenesis through osteoimmunomodulation [132]. In another study, Zhu et al. reported that a honeycomb-like titania surface with a groove diameter of 3,948 ± 282 nm activated RAW 264.7 cells to polarize into the M2-phenotype via Rho family protein (RhoA, Rac1, and CDC42) signaling and facilitate the secretion of various anti-inflammatory cytokines, such as IL-4 and IL-10 [9]. This study also demonstrated that a TiO₂ nano-groove with a diameter of at least 90 nm was sufficient to stimulate filopodia development. The RAW 264.7 cells with M2 phenotype (grown on titania 3,948 nm structure) were found to stimulate BMP-2 release from MSCs by enhancing osteoimmunomodulation. Similarly, Leong et al. reported that PCL/PLA/PDMS-based micro/nanoimprinted surfaces with varying groove diameters regulate the phenotype of RAW 264.7 cells by controlling adhesion and morphology (Fig. 4). This study also found that in the control (flat 2D surface) samples, the macrophage morphology was oval-shaped and with fewer filopodia [129]. They were positive for TNF-α, a common marker of the M1 phenotype. In contrast, the RAW 264.7 cells were found to exhibit a spindle-shaped morphology with elongated filopodia in the presence of a PDMS stamp with 500 nm and 2 μm PCL/PA micro/nano patterning. They were also positive for VEGF, a common intracellular marker of the M2 phenotype. Table 4 depicts an overview of various micro/nano topology-guided macrophage polarization behaviors and possible tissue engineering applications. Therefore, surface topology greatly influences the morphological changes in macrophages and the differential secretion of cytokines due to the foreign body reaction. Apart from micro/nano topology, electrospun nanofibers have less inflammatory response and mild foreign reaction with immune cells owing to less foreign body reaction [133]. In conclusion, biomaterials with varying surface topologies have a potential influence on the polarization behavior of immune cells and could be used as ideal immunomodulatory substrates for tissue engineering. Moreover, the tissue/organ-mimicking topographical structures should be developed in the future for facilitating dynamic polarization of macrophage cells when the implantable biomaterial stays longer time in the body. Taken together, topographical cues and tissue/organ-mimicking nanostructures has potential role macrophage polarization in tissue engineering.

Fig. 4.

Effect of micro/nano polydimethylsiloxane (PDMS) patterning on macrophage polarization. (a & b) Field-emission scanning electron microscopy (FE-SEM) images of RAW 264.7 cells growing on 2 μm PDMS pattern and flat PDMS surface, respectively, showing the variation in cell morphology. (c & d) Fluorescence (FL) and bright-field microscopy images of M2 polarized RAW 264.7 cells, showing the direction of growth in the presence of 2 μm PDMS pattern. (e–j) FL microscopy images of RAW 264.7 cells, showing the F-actin (green) distribution in the presence of various coated substrates. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars: 20 and 50 μm [129].

Table 4.

Examples of surface topology-guided macrophage/monocyte activation and its potential application in tissue engineering and regenerative medicine.

Topological cues	Type of immune cells	Immunomodulatory effects	Applications	References
Aligned PCL nanofiber with grooved surface	RAW 264.7	Activation of pro-inflammatory phenotype, IL-1β and TNF-α secretion	Tendon regeneration	[134]
Ti substrate with 5 μm groove	BMDM	Activation of anti-inflammatory phenotype, enhanced IL-10 secretion	Wound healing	[6]
Surface engineered Zn micro/nano pattern	THP-1	Activation of anti-inflammatory phenotype, enhanced CCR-7 and CD209 expression	Macrophage-assisted osteoinduction and enhanced osteogenic differentiation	[7]
Aligned PLCL nanofibers	RAW 264.7	Activation of pro-inflammatory phenotype	Peripheral nerve regeneration	[135]
Commercial surgical gauge	RAW 264.7	Activation of anti-inflammatory phenotype, enhanced expression of CCR7 and CD206	Myogenesis	[136]
3D printed porous titania modified with PEO	hMDM	Activation of anti-inflammatory phenotype, enhanced secretion of IL-10 and CD163	Enhancing osseointegration	[137]
Glycosaminoglycan functionalized aligned collagen fibers	Human primary monocytes	Activation of anti-inflammatory phenotype, enhanced expression of IL-10	Wound healing	[138]
PDA@TiO2 coated with Sr2+ and Ag2+ metals	RAW 264.7	Activation of anti-inflammatory phenotype, enhanced expression of CD206 marker	Macrophage-assisted osteoinduction and enhanced osteogenic differentiation	[139]
Tanshinone-loaded aligned PCL nanofiber (1 μM Tan-PCL)	RAW 264.7	Activation of anti-inflammatory phenotype, Enhanced expression of Arg-1, Fizz-1, and Ym1	Enhancing angiogenesis	[140]

RAW 264.7 = murine macrophage cells; BMDM = human bone marrow-derived macrophages; THP-1 = human primary monocyte cells; hMDM = human monocyte-derived macrophages.

3.2. Effect of biochemical properties on macrophage fate

3.2.1. Material composition

Biomaterial composition is another important factor affecting the immunomodulation of macrophages. For example, biomaterials composed of silicate, silicate/phosphate, bioactive glass, bone ceramics (calcium phosphate), and metal ion-doped polymers have been designed in the form of a scaffold, printable hydrogel, thin film, coating, or fiber, which undergo time-dependent degradation and their degradation products may activate immune cells in various forms [141]. After the degradation of a biomaterial scaffold, various bioactive components, such as ions and proteins, are released in the body fluid, triggering a local immune response. Recently, various strategies have been employed to engineer bioactive scaffolds through multiple polymer grafting or surface functionalization by incorporating various functional groups. When bioengineered scaffolds are implanted at a surgical site, they effectively modulate macrophage polarization and promote the secretion of various cytokines and chemokines [28,142,143]. In a recent study, a silk fibroin/polypropylene-based scaffold showed a remarkable increase in local immune response when transplanted in vivo. The polypropylene effectively stimulated macrophages via foreign body reaction, and increased Th1 and cytotoxic T cells (T_c) were found to accumulate at the implantation site after 3 weeks [144]. The nature of biopolymers (synthetic or natural) also determines the type of immune response in the body. Synthetic or chemically derived biopolymers (e.g., polyethylene glycol) mostly interact with neutrophils and initiate a chronic inflammatory response, followed by the activation of pro-inflammatory signals. Naturally derived polymers have been found to stimulate the activation of Arg-1, Chil3, Gata3, and CD163 [145]. In contrast, naturally derived polymers (e.g., gelatin, collagen, chitosan, and other bioactive components) may trigger anti-inflammatory (tissue-healing response) signals in the human body. Cha et al. showed that IL-4-loaded gelatin-methacryloyl (GelMA) and polyethylene glycol diacrylate (PEGDA) hydrogel activated M2 polarization of THP-1 cells via α2β1-integrin protein (a type of protein present in the cell membrane) signaling [146]. The authors also demonstrated that the composite hydrogel stimulated the expression of STAT6 and IL-10 and downregulated the expression of IRF5 and IL-6. Thus, the GelMA/PEGDA hydrogel is thought to promote M2 polarization via the α2β1-integrin/STAT6 signaling axis. Table 5 depicts an overview of how material composition regulates macrophage fate during the immune response. Although several reports have demonstrated the potential role of various biomaterials in macrophage polarization, extensive research on the polarization mechanism must be conducted before actual clinical application. Furthermore, the composition of the bioceramic material is also crucial for the immune response. For example, when applying one or two metal ions or a series of various bioactive ions, it is difficult to identify which ionic gradient affects (pH or salinity) the polarization potential of macrophages. Therefore, the proper choice of bioceramic or polymer scaffold should be considered before successful implantation.

Table 5.

Effect of biomaterial compositions on monocyte/macrophage polarization.

Biomaterial compositions	Components	Type of immune cells	Nature of immunomodulation	References
Inorganic materials	Ca2+	Primary monocytes	Ca2+/Wnt-mediated signaling and enhanced inflammatory response	[147]
	Co2+	RAW 264.7	Co/TiO2 matrix promoted M1 macrophage polarization and phagocytosis-mediated enhanced bactericidal efficacy	[148]
	Cu2+	RAW 264.7	Cu-doped SPEEK material promoted rapid bacteria clearance through M1 macrophage polarization; Cu-doped mesoporous silica nanoparticle enhanced osteoclastogenesis through M2 macrophage polarization	[149,150]
	Fe3+	Human primary macrophages	Concentration-dependent M1 phenotype activation of macrophages and pro-inflammatory effects	[151]
	Li+	BMDMs	Li-based bone ceramics modulate in vivo osteoclastogenesis via M2 macrophage polarization	[152]
	Mg2+	Human primary monocytes; RAW 264.7	Mg-based (MgSO4) nanocomposite promoted anti-inflammatory phenotype and decreasing the secretion of IL-6 and TNF-α; MgSiO3-based composite exhibited better osteoclastogenic activity via macrophage immunomodulation; MgO NPs confers the M1 phenotypic switch of RAW 264.7 cells and induced osteogenesis	[[153], [154], [155]]
	Sr2+	RAW 264.7	Sr-coated bioactive glass induced TRAP-medicated osteoclastogenesis	[156]
	Zn2+	RAW 264.7	ZnO nanocomposite films activated M1 macrophages and induced phagocytotic bacteria killing; Zinc silicate/calcium phosphate scaffold promoted M2 macrophage polarization and immunomodulation-assisted enhanced osseointegration	[156,157]
	Se4+	RAW 264.7	Se NPs coated TNTs exhibited anti-inflammatory function of RAW 264.7 cells and broad-spectrum anti-bacterial activity	[158]
	MoO4−	BMDMs	Sustained release of MoO4− from Mo-bioactive glass scaffold induced M2 macrophage polarization via upregulation of Arg, CD206, and IL-4 production	[159]
Surface functional group (anionic)	Sulfonate (-SO3H)	RAW 264.7	Nitro and sulfonic acid moieties present in poly(N-isopropylacrylamide-co-acrylic acid) hydrogel stimulated M1 polarization of macrophages, while the amide containing hydrogel induced M2 polarization	[160]
	Amine/Carboxyl (-NH2/-COO-)	Human primary macrophages	Stimulated the early expression of pro-inflammatory factors (M1) and later induce the anti-inflammatory factors (TGF-β and mATP) secretion in M2 polarized macrophages	[110]
	Amine (-NH2)	RAW 264.7; BMDMs	Amine-modified bioactive glass promoted M2 polarization of macrophages and increased the production of Arg-1 and IL-10	[161]
	Guanidinium (CH6N3+)	RAW 264.7	Polarization towards M1 axis and stimulated the production of fibrotic scar at the wounded site	[162]
Surface functional group (cationic)	Polyethyleneimine (PEI)	THP-1; RAW 264.7	Cationic super paramagnetic iron oxide nanoparticles (PEI-SPIONs) induced the M1 polarization of macrophages	[163]
Membrane-derived proteins	Integrins	BMDMs	Knockout macrophage-1 antigen (Mac-1) may regulate the polarization towards M1 axis and accelerate the fibrotic scar formation	[164]
Growth factor/cytokines	IL-4	RAW 264.7; BMDMs	IL-4 incorporated biomaterials stimulated M2 polarization of macrophages for tissue regeneration; IL-4 coated/PDA incorporated TiO2 boosted M2 polarization and facilitated soft tissue regeneration in vivo; sustained release of IL-4 from stiff gelatin matrix promoted M2 macrophage polarization and enhanced osteogenic differentiation	[[165], [166], [167]]
	IL-13	RAW 264.7	Direct application of IL-4 inhibited atherosclerotic plug clearance via enhancing the production of anti-inflammatory macrophages	[168]
	(IL-4 + IL-10 + TGF-β) cocktail	Human primary monocytes	Multiple application of Th2 factors may trigger the M2 polarization and enhanced anti-inflammatory factors secretion	[169]
	IFN and IL-4	Human primary macrophages	Controlled release of IFN-γ/IL-4 from d-ECM scaffold triggered the M2 macrophage polarization and enhanced the new blood vessel formation (neo-angiogenesis)	[170]
	IL-6	Murine adipose-derived macrophages	IL-6 administration boosted the activity of Th2 factors which in turn triggered the M2 polarization of macrophages during obesity	[171]
Decellularized extracellular matrices (d-ECMs)	Porcine d-ECM	RAW 264.7	d-ECM scaffold promoted M2 phenotype of murine macrophages	[172]
	Urinary bladder ECM (ub-ECM)	RAW 264.7	Initiated type-2 immune response and inhibited tumor metastasis via secretion of pro-inflammatory cytokines for cancer immunotherapy	[173]
	Heart-derived ECM (c-ECM)	RAW 264.7	Cardiac d-ECM scaffolds boosted the activation of M1 phenotype and induced the fibrotic scar formation	[174]
Organoids/Spheroids/Cell sheet/enzymes	Human dermal fibroblast (hDF)-derived biomaterials	RAW 264.7	Macrophage polarization towards M2 axis and enhanced angiogenic response for wound healing application	[175]
	Lysyl-tRNA synthetase	THP-1	Induced the production of anti-inflammatory cytokines and promoted tumor metastasis (tumor associated neo-angiogenesis)	[176]

3.2.2. Material degradation

After implantation of a biomaterial scaffold, it shows time-dependent degradation owing to the activity of various enzymes present in the body fluid. The degradation of a biomaterial scaffold leads to a shift in surface topography, changes in chemical composition, and stiffness, which attracts immune cells to differentiate or polarize [141]. For example, controlled degradation of β-tricalcium phosphate (β-TCP) has shown a significant amount of Ca²⁺ release, boosting the M2 polarization of dendritic macrophages via signaling through the calcium-sensing receptor (CaR). CaR stimulates the production of anti-inflammatory cytokines, which enhances the production of BMP-2 in MSCs during osteogenesis [177]. In another study, a biphasic calcium phosphate scaffold was found to stimulate the M2-like phenotype of RAW 264.7, during in vitro immunomodulation. In contrast, non-biodegradable biomaterials (e.g., chemically crosslinked polymers or thermoplastic polymers) exhibit serious inflammatory reactions and induce the fibrotic process (delayed wound healing) owing to the recruitment of M1 pro-inflammatory macrophages [136]. Therefore, the selection of appropriate bioactive but biodegradable polymers may contribute to selective immunomodulation, which is crucial for in vivo tissue regeneration.

3.2.3. Soluble factors

Various soluble factors have been found to stimulate macrophage differentiation. Macrophages can be polarized into M1 or M2 macrophages via the direct application of soluble growth factors or cytokines. Growth factors, such as BMP-2 [[178], [179], [180], [181]], VEGF [111,[181], [182], [183]], plasminogen activator inhibitor-1 (PAI-1) [184,185], epidermal growth factor (EGF) [186], and fibroblast growth factor (FGF) [186], and cytokines, such as IL-4 [187,188] and IL-10 [189,190], have been shown to induce inflammatory responses in human dendritic monocytes/murine macrophages. During acute burns or trauma, the skin tissue is affected by various air-borne pathogens, which initiate a local immune response known as pathogen-associated molecular patterns (PAMPs). At this stage, blood monocytes accumulate in the wounded region and secrete pro-inflammatory cytokines to combat pathogens. Approximately 2 weeks after primary wound healing, a number of anti-inflammatory cytokines (e.g., IL-4, IL-10, and IL-13) have been found to accumulate at or near the w ound bed, resulting in rapid proliferation and differentiation of fibroblast cells [191]. Biomaterial scaffolds can be used for sustained delivery of various growth factors/cytokines for tissue regeneration. Kwon et al. reported that mesoporous silica nanoparticles (MSN) loaded with IL-4 resulted in M2 macrophage polarization and upregulated the expression of Arg-1 and Chil3 transcription factors in BMDMs [192]. It was also demonstrated that larger pores (∼180 nm) on the surface of silica are responsible for higher IL-4 loading and greater M2 polarization efficiency in BMDMs. In another study, PCL/polyvinyl alcohol (PCLPVA) nanofibers modified with BMP-2 promoted prolonged M2 polarization of macrophages and vascularized bone regeneration via immunomodulation [193]. The controlled delivery of BMP-2 from the core-shell nanofiber facilitated higher bone regeneration efficiency (∼76.38 ± 4.13%) through rapid vascularization and local immune response in a calvaria defect model. Owing to the advantages of soluble factors that trigger local immunity, the optimum concentration (e.g., ng or nM or pM) or selectivity of the soluble factors (e.g., factors with one type or a combination of various factors) should also be considered. Furthermore, a thorough understanding of the soluble factors, their chemical structure, and proper immunomodulation mechanisms is highly desirable for successful clinical application.

3.2.4. Other stimuli-assisted platforms

Macrophage polarization can be tailored using various biophysical (e.g., electric, magnetic, light, and ultrasound) or biochemical (e.g., pH and gas flow) stimuli in vivo. Biophysical or biochemical stimuli create a dynamic microenvironment for immune cells and direct specific types of polarization based on the nature of stimulation. Bian et al. showed that non-invasive magnetic stimulation through arginine-glycyl-aspartic acid (RGD ligand)-modified superparamagnetic iron oxide nanoparticles (SPIONs) promoted RAW 264.7 cell polarization (Fig. 5a) both in vitro and in vivo [194]. Remote oscillation of SPIONs provided a dynamic environment in the mouse subcutaneous wound and promoted macrophage polarization during tissue regeneration (Fig. 5b). Magnetic fields can also alter calcium homeostasis and actin reorientation in monocytes and facilitate the polarization toward M1 or M2 phenotype (Fig. 5c). For example, Wosik et al. found that uneven magnetic field treatment caused rapid elongation of F-actin (a cytoskeletal protein) and induced polarization of macrophages (Fig. 5d) toward the M2 axis [195]. This study also revealed that RhoA protein plays a critical role in the magnetic movement of cytoskeletal proteins. Compared to control macrophages (RhoA^+/+; wild type), mutant macrophages (RhoA^−/−) exhibited early accumulation of actin stress fibers and disruption of actin fibers under prolonged exposure to a magnetic field, which was mainly regulated by the Ca²⁺/RhoA complex (Fig. 5e and f).

Fig. 5.

Biophysical cues regulating the monocyte/macrophage fate. (a–c) Schematic illustration of the arginine-glycyl-aspartic acid (RGD)-modified superparamagnetic iron oxide nanoparticles (SPIONs) regulating the macrophage polarization under varying frequency range [194]. FE-SEM and atomic force microscopy images showing the morphology of the fabricated SPIONs and their magnetic field-guided oscillatory movement. The oscillatory behavior of the RGD-SPIONs promoted M1 and M2 polarization under high- and low-frequency vibrations, respectively. Scale bar: 50 nm, 1 μm, and 50 μm [194]. (d–f) Effect of magnetic field stimulation on the distribution of focal adhesion proteins (vinculin and RhoA). The magnetic field (1.24 T) treated macrophages exhibited an increase in podosome production. FL microscopy images of M2 polarized macrophages, showing the expression of TRMP2 protein. Schematic diagram of RhoA-dependent induction of macrophages under magnetic field stimulation. Scale bar: 50 μm [195]. (g, h) Schematic diagram of the light-responsive hyaluronic acid modified with RGD peptides for immunomodulation. UV light exposure significantly enhanced the expression of intracellular Arg-1 production through periodic activation of αvβ3 integrin protein. Scale bar: 30 μm [196].

In addition, electric and magnetic fields have different effects on the macrophages. Recently, several studies have demonstrated that near-infrared light significantly manipulated macrophage migration and polarization properties both in vitro and in vivo. For example, Chen and colleagues showed that a light-responsive hyaluronic acid polymer facilitated the controlled release of RGD adhesion peptides and allowed αvβ3 integrin to bind with the polymer to modulate the immune response [196]. Thus, macrophage polarization can be augmented by various photo-responsive polymers during tissue regeneration. Human skin is electrically conductive, and its electric potential changes under varying physiological conditions, such as injury or trauma. A low voltage-frequency electric field (3 V-1 Hz, 20 min/day treatment) has been shown to promote osteogenesis [197] and allow macrophage cells to differentiate into various phenotypes [198]. Moreover, radiotherapy was found to promote pro-inflammatory activation of TAMs owing to the greater expression of inducible nitric oxide synthase (iNOS); therefore, it is ideal for cancer immunotherapy [199]. Although several biophysical or biochemical stimuli-based platforms have shown promising biomedical applications, little progress has been made in understanding the molecular mechanism behind cell-stimuli interactions. Future research should be conducted using Food and Drug Administration (FDA)-approved stimulation doses and monocyte/macrophage cell lines from various sources (e.g., human, rat, and mouse), with a detailed emphasis on the molecular activation and physiological response during the immune response. Finally, extensive in vitro and in vivo studies should be conducted to validate the clinical application of physical stimuli-based platforms. Fig. 5 illustrates the use of various stimuli-responsive biomaterials for macrophage immunomodulation.

4. Molecular mechanisms of biomaterial-macrophage interaction

As discussed earlier, macrophages respond in various ways upon contact with biomaterial surfaces. The biomaterial property greatly influences the heterogenicity of immune activation in both in vitro and in vivo systems. The macrophages initiate foreign body reaction (FBR) after culturing cells in a tissue culture plate or in vivo system. Next, various proteins (e.g., serum albumen, fibrinogen, fibronectin, vitronectin, and immunoglobulins) from the blood and intestinal fluid accumulated on the biomaterial surface, forming a thick proteinaceous coat or layer. In addition, platelets in contact with biomaterial surface release various chemoattractant signals, ensuring the migration of immune cells and fibronectin proteins to form a primary ECM [[200], [201], [202]]. Long-term implantation of biomaterial may induce an acute inflammatory response in our body. The M1 macrophages secrete various pro-inflammatory factors, ROS, and degradative enzymes that influence the macrophages to fuse. After fusion, a large cell mass is generated, known as foreign body giant cells (FBGCs) (Fig. 6). At a later stage, the M2 macrophages migrate to the wound site and induce fibrotic scar formation owing to the activity of VEGF and TGF-β1 via cross-talk with fibroblasts. The primary ECM acts as a recognition site for recruited macrophages or resident macrophages to induce the secretion of various cytokines and chemokines [203].

Fig. 6.

Schematic illustration of the host response to biomaterials during wound healing and regeneration [203].

Evidence also demonstrated the involvement of activation of various inflammasomes during macrophage-assisted FBR. Inflammasomes act as signal transducing for initiating a series of molecular events in macrophage cells. Inflammasomes are large cytoplasmic protein complexes having the ability to recognize pathogen-associated molecular patterns (PAMPs), danger-associated molecular patterns (DAMPs), and lifestyle-associated molecular patterns (LAMPs). To gain more insightful knowledge about inflammasomes, a reader is encouraged to study the cited literature [[204], [205], [206]]. The inflammasomes are primarily activated through the membrane-bound receptor proteins, known as toll-like receptors (TLRs). The signal transduction process in macrophages involving the activation of the M1 or M2 phenotype depends on the nature of the biomaterials. For example, the nanoparticles or nanocrystals with a diameter of less than 100 nm are reported to internalize by the macrophages [207], whereas the biomaterial scaffold or micro/nano-patterned surface induces the mechanobiological stimulation to macrophages during immunomodulation [9]. This section briefly summarizes the various macrophage polarization signaling mechanisms in response to multiple biomaterials and/or nanotopographical structures.

4.1. Inflammasome activation and NLPR3-mediated signaling

Various nanomaterials have been shown to promote TLR/NLPR3-mediated inflammasome activation in resident macrophages. The inflammasome activation is directly connected to the processing of IL-1β after being contacted or engulfed nanoparticles (Fig. 7a). A study conducted by Maitra et al. [208] showed that polyethylene microparticles and alkane biopolymers might induce the activation of NLPR3 inflammasome-mediated activation of pro- IL-1β and subsequent M1 polarization of macrophages. In another study, Bueter et al. [209] reported that chitosan effectively triggers the inflammasomes of macrophages by activating intracellular K⁺ ions, reactive oxygen species (ROS), and lysosomal destabilization. This study used various inflammasome inhibitors, demonstrating the downregulation of different inflammasome-associated markers. Similarly, polymethyl methacrylate (PMMA) microspheres induced a higher expression of IL-1β with Caspase-1, NLPR3, or ASC when injected in a subcutaneous mice wound model [210]. This study also demonstrated that mutant mice deficient in inflammasome-mediated marker (nrlp3^−/−) had reduced expression of IL-1β or NLRP3 or Caspase-1, suggesting that inflammasome generation played a crucial role in M1 macrophage polarization.

Fig. 7.

Molecular mechanisms of biomaterial-macrophage interaction. (a) NLPR3-mediated signaling, (b) MCP-1/Rac1-mediated signaling, (c, d) RhoA/Rac-mediated cytoskeletal signaling [9,212], (e) TNF/NF-kB signaling, and (f) JAK/STAT-mediated signaling in macrophages.

4.2. MCP-1/Rac1-mediated signaling

MCP1 and Rac1-mediated signaling plays a vital role in macrophage polarization (Fig. 7b). Several studies indicated that phagocytic macrophages underwent MCP-1 and Rac1-mediated cytoskeletal remodeling with phosphatidyl serine (PtdSer) and subsequent activation of CD36. A study by Valles et al. [211] showed that THP-1 cells expressed a high level of TNF-α and MCP-1 when cultured in a 2D polystyrene scaffold up to 72 h of incubation. However, after 96 h, the expression of TNF-α and MCP-1 was downregulated in 2D culture, suggesting that the THP-1 cells were in a reduced inflammatory stage. This study also showed that 3D scaffold promoted reduced expression of TNF-α, whereas long-term incubation promoted MCP-1 dependent TNF-α expression. In another study, the MCP-1 knockout mice (mcp-1^−/−) exhibited reduced FBR, owing to the low secretion of TNF-α, suggesting the role of MCP-1 is involved in TNF-α mediated immune response in vivo [213]. Furthermore, IL-4-induced MCP1-KO mice were found to have defective macrophage fusion with normal E-cadherin (E-cad) and β-catenin expression. The reduced expression of E-cad and β-catenin were also associated with Rac1 and MMP-9 expression, which regulate a balance between MCP-1 and Rac1 expression [214].

4.3. RhoA/Rac-mediated cytoskeletal signaling

Recently, focal adhesion proteins have been found to regulate macrophage polarization in response to the 3D micro/nano topographical scaffolds (Fig. 7c). In this context, superhydrophilic titania nanosurfaces with dense nanospikes promoted M1 macrophage polarization through selective binding with focal adhesion kinases (FAK) and TLR2/4. The FAK signaling induced MACRO/SR-A mediated signaling cascades, resulting in the early expression of TNF-α and iNOS [212]. In another study, TiO₂ honeycomb groove with a 90 μm diameter was found to induce the RhoA/ROCK signaling during macrophage polarization [9]. This study also demonstrated that TiO₂ nanostructure with varying diameters facilitated the integrin-β₁ binding of macrophages and which promoted the binding of Src/Paxillin/Vinculin complexes (Fig. 7d). The integrin-β₁ was found to activate the Rac1/RhoA/CDC42 complex and facilitated the filopodia or podosome formation. The study further demonstrated that filopodia formation and RhoA/ROCK signaling involved M1/M2 polarization. The TiO₂ structure with 90–500 μm pores induced the RhoA/ROCK-mediated PPAR signaling and upregulation of M2 markers, whereas, TiO₂ with 1000–5000 μm structure was found to influence the TNF-α signaling and overexpression of M1 markers in macrophages. Thus, RhoA/Rac-mediated cytoskeletal rearrangement responding to biomaterial surface is critical for macrophage polarization.

4.4. TNF/NF-kB-mediated signaling

TNF/NF-kB signaling is another important signaling pathway modulating macrophage polarization. Studies indicated that the intracellular TNF level is crucial for activating the NF-kB transducing element in both in vitro and in vivo models, thus conferring the development of novel immunomodulatory biomaterials [215,216]. Various topographical nanostructures have been shown to promote the M1 polarization of macrophages owing to the overexpression of TNF-α. TNFR is a potential inducer of NF-kB during FBR (Fig. 7e). In the absence of TNF-α, the p50, and p65, the canonical component of NF-kB remains inhibited by another element IkB. During FBR, the TNF-α is activated by TNFR or TLR and induces the TAK1, a cytoplasmic inducer of TNF. The activated TAK1 and TNF further promoted the aggregation of IKK hetero-dimer, which later induced the phosphorylation of IkB. The phosphorylated IkB binds with the p50/p65 and forms a trimeric complex. The trimer is later transported into the nucleus and induces the NF-kB element and pro-inflammatory gene transcription. In contrast, the non-canonical NF-kB signaling is mediated through NIK-dependent induction of IKK via phosphorylation of p100 and subsequent formation of the p52-RelB complex. The p50/p65-mediated NF-kB pathway is usually observed during in vivo implantation of titania and copper-based biomaterials [217]. Studies also confirmed that both the canonical and non-canonical pathways actively participate during IL-4-induced immune reaction via macrophage fusion (in vitro and in vivo) or osteoclast fusion (in vivo) [218,219]. Taken together, these reports confirmed that TNF/NF-kB signaling is essential during FBR-mediated macrophage polarization.

4.5. Wnt/β-catenin-mediated signaling

Recent studies also indicated that Wnt signaling plays a vital role in macrophage proliferation and differentiation during FBR-mediated immune response both in vitro and in vivo. Macrophage Wnt signaling initiates innate immune response, critical for tissue regeneration. Thus, inhibition or loss of Wnt function may result in fibrotic symptoms owing to the loss of activity of MMPs in both in vitro and in vivo models [[220], [221], [222]]. Wnt signaling is usually connected to the bone-marrow macrophages as it plays a crucial role in osseointegration [223]. A study conducted by Abaricia et al. [224] demonstrated that biomaterial surface properties, such as roughness and hydrophilicity, induce M2 macrophage polarization via inducing Wnt signaling. This study also emphasized that macrophages in Wnt mutant mice (Wnt^−/−) showed reduced inflammatory properties owing to the reduced activity of IL-6, IL-12, TNF-α, and CXCL10 when implanted with titania-based biomaterials. The wild-type mice showed high expression of Wnt family genes (Wnt1, Wnt2, Wnt3, Wnt4, Wnt5a, and Wnt5b) in mice, with a high level of TNF-α and IL-6, indicating the role of Wnt signaling in macrophage-mediated disease progression. Thus, understanding the Wnt signaling pathway of macrophages will be beneficial for developing next-generation immunomodulatory biomaterials.

4.6. JAK/STAT-mediated signaling

Macrophage fusion is usually associated with JAK/STAT signaling pathway during FBR. It has been shown that IL-4 induction promotes macrophage fusion and activates JAK/STAT signaling cascades. The IL-4 induction triggers the JAK1/3 and STAT6 or MCP-1, leading to the upregulation of β-catenin and E-cad in M1 macrophages. Patel el al [207] recently reported that rod and spherical nanocellulose (r-CNC and s-CNC) might trigger M1 polarization of RAW 264.7 cells via STAT1 signaling pathway while downregulating the NF-kB signaling pathway and inducing the expression of TNF-α, iNOS, and CD68 gene markers after 24 h of treatment in vitro. These results suggest that JAK/STAT signaling positively influences macrophage polarization in respond to biomaterials.

5. Host response to biomaterials in vivo

After the implantation of a biomaterial scaffold (foreign body), migration of both Ly6C⁻ and Ly6C⁺ macrophages increased at the biomaterial site. The increased mobility of Ly6C⁺ monocytes enhanced the proliferation of tissue-resident macrophages. During mild inflammation, the tissue-resident macrophages proliferate rapidly [58,225]. However, the circulating monocytes were found in higher numbers during the acute inflammatory response (Fig. 8a). The circulating monocytes rapidly transformed into monocyte-derived macrophages and arranged themselves around the implanted biomaterial (Fig. 8b). Studies indicate that in mice, a weak de novo expression of CD11b, increased CD11b, and decreased CD45, CD68, CD71, CD86, and CD206 suggested a transition from monocyte to macrophage after biomaterial implantation [58]. The exact phenotype of macrophages in vivo is difficult to identify because both monocyte and macrophages have similar types of markers. Thus, most in vivo macrophage analysis is restricted to only polarization studies. A study conducted by Liu et al. reported that macrophages in response to a 3D hetero-nanostructured scaffold displayed positive for CD68 (M1 marker) and CD206 (M2 marker) after 4 weeks’ post-implantation (Fig. 8c). This study also indicated that long-term scaffold implantation might promote the higher expression of CD206 than CD68, suggesting tissue healing and regeneration [226]. Similarly, Wang et al. reported that surface-functionalized TiO₂ bone implants showed variable expression of CD68 and CD206 in vivo. The expression of CD68 was higher in pure TiO₂ and DBCO-modified TiO₂. However, the CD68 expression was drastically decreased after incorporating Zn, BMP-2, or Zn/BMP-2 modification, suggesting that M1 polarization was only dominant in the early phase of biomaterial implantation during bone regeneration (Fig. 8d). Following that, the expression of IL-10 (anti-inflammatory cytokine) was significantly increased after 4 weeks post-implantation of Zn/BMP-2 modified titania scaffold [227]. These two studies indicate that proper modification of biomaterials with various nanomaterials or proteins may induce M1 polarization of macrophages immediately after implantation owing to FBR and M2 polarization after 2–4 weeks. 3D-printed PCL/nHAp scaffolds have long been explored as an ideal bone implant for in vivo bone regeneration. The glycopeptide-modified PCL/nHAp scaffold (Fig. 8e) demonstrated strong evidence of M2 macrophage polarization during in vivo bone regeneration by enhancing the expression of the CD206 marker [228]. It is also noticed that the higher expression of the CD206 marker positively correlates with osteogenic markers expression (e.g., Runx2), further suggesting that M2 polarization plays a crucial role tissue healing and regeneration.

Fig. 8.

(a, b) Histological identification of circulating monocyte (arrow) obtained after Pappenheim staining. Accumulation of macrophages (arrow) around the implanted biomaterial [58]. (c–e) Expression of M1 (CD68 and CD86) and M2 (CD206 and IL-10) macrophage markers during bone regeneration in vivo [[226], [227], [228]].

6. Tissue-specific biomaterials for immunoengineering

As discussed earlier, monocytes/macrophages reside in various tissues, such as skin, muscle, liver, heart, spleen, lung, kidney, eye, and bone. Based on the localization, the macrophages play essential roles in healing and regeneration of various organs. Recently, extensive research has been devoted to studying the effects of biomaterials on macrophage polarization and their positive feedback on tissue healing and regeneration. During wound healing, macrophages are polarized to the M1 phenotype to combat pathogenic attacks. However, at later stages, M2 macrophages accumulated in the wound site, promoting fibroblast maturation, blood vessel formation, and skin re-epithelialization. Based on their function and activation, cytokines that play major roles in tissue remodeling are IFN-γ, TNF-α, IL-17, MCP-1, IL-10, IL-13, IL-6, EGF, FGF, TGF-, PDGF, and VEGF. M2 macrophages promote growth factor secretion, angiogenesis, cell proliferation, and ECM assembly to accelerate tissue regeneration. Therefore, biomaterials with excellent immunomodulatory properties, low cytotoxicity, and exceptional antimicrobial properties are highly desirable for wound-healing applications. Similarly, bioceramic-based bone grafts play an important role in immunopolarization by recognizing the body's immune system and facilitating the desired immune reaction. An ideal bone graft must have desirable mechanical strength, porosity, particle size, and mineral ions to boost osteogenesis and osteoimmunity. Surface wettability and topography significantly affect bone development and trigger the production of various cytokines (IL-6 and IL-1β) and chemokines (IL-8 and RANTES), epithelial-derived neutrophil-activating peptide-78 (ENA-78, also known as CXCL5), and monocyte chemoattractant protein-1 (MCP-1) [229,230]. The porous nature of the bone implants may affect the immune response and subsequent osteoimmunomodulation [231,232]. Implants with small pores may hinder the diffusion of nutrients and oxygen from the blood and body fluids (interstitial fluid), creating a local hypoxic microenvironment [233]. Local hypoxia leads to the activation of M1 macrophages, ultimately leading to granuloma formation. Additionally, local hypoxia may trigger the bone cells to release hypoxia-inducible factor-1α (HIF-1α), which promotes angiogenesis. The induction of HIF-1α and neo-angiogenesis is beneficial for bone regeneration. Previous studies have demonstrated that biomaterial scaffolds with moderate porosity (∼90–120 μm) hinder angiogenesis while triggering chondrogenesis. In addition, biomaterial scaffolds with larger pores (∼220–350 μm) may induce vascularization and osteogenesis by enhancing nutrient exchange and oxygen diffusion [[234], [235], [236], [237], [238], [239], [240]]. Table 6 depicts an overview of the biomaterial scaffold-induced immunomodulation of macrophages for tissue healing and regeneration.

Table 6.

Biomaterial scaffold-guided immunomodulation for tissue healing and regeneration.

Tissue/organ	Biomaterial	Immunomodulatory functions	References
Bone	Cerium oxide NPs/TiO2 nanocomposite	Ce4+/Ce3+ exhibited concentration dependent M2 polarization of macrophages and promoted bone regeneration	[154]
	γ-Fe2O3/HAp/PLA scaffold	Magnetic nanoparticles stimulated the mechanosensing responsive factors and boosted M2 polarization of macrophages, induced bone mineralization, and vascularized bone formation	[241]
	Al2O3 nanoporous scaffold	Al2+ exhibited size-dependent macrophage phenotyping and directed anti-inflammatory cytokines secretion for bone regeneration	[242]
	Alginate/Tyramine/Sericin/GO-based injectable hydrogel	Sericin/GO induced M2 polarization via NF-kB and MAPK signaling pathway and induced osteogenesis in rat BMSCs; accelerated irregular bone defect in a distal femur defect model	[243]
	Silicified gelatin/polyacrylamide nanocomposite	Promoted M2 macrophage polarization and induced vascularized bone regeneration in a calvaria defect model	[244]
	Fe2O3/PEG/collagen nanocomposite scaffold	Magnetically guided M2 polarization and robust bone regeneration; the scaffold induced the secretion of IL-10 and promoted differentiation of BMSCs	[245]
	Piezoresistive Ti3C2/SF nanocomposite hydrogel	The Ti3C2 boosted M2 macrophage polarization; secreted IL-4; induced the angiogenesis and osteogenesis via immunomodulation; the macrophage conditioned media activated Ca2+/CAM signaling in osteoblast during osteogenesis	[246]
Skin	PEG/heparin/PCL scaffold	Anti-inflammatory activation (MCP-1, MIP-1α, MIP-1β, and IL-8) of macrophages and promote wound healing in a subcutaneous wound model	[247]
	SF/HA/PCL scaffold	HA induced the M1 phenotype of macrophage, inhibited protein absorption, and promoted fibrotic scar formation	[248]
	FTY720-doped PLGA scaffold	Repaired skin injury via activation of M2 macrophages and boosted the wound healing process	[249]
Nerve	Cerium oxide NPs	Decreased ROS level and recovered from spinal cord injury	[250]
Muscle	ECM-derived bioscaffold	The enzymatic degradation product of bioscaffold induced M2 macrophage polarization and favored muscle tissue regeneration	[251]
Blood vessel	PCL scaffold	PCL scaffold with larger pore (∼30 μm) promoted M2 polarization and enhanced the angiogenesis	[252]
Heart	Decellularized cardiac ECM scaffold	Decellularized pericardium scaffold promoted anti-inflammatory activation of immune cells and sustained the regeneration capability of cardiomyocytes	[174]

7. 3D bioprinting and immunoengineering for tissue regeneration

3D bioprinting is an emerging AM technique that surpasses the existing barriers in tissue engineering and regenerative medicine. The key principles of 3D bioprinting rely on three basic concepts: (1) biomimicry, (2) self-assembly, and (3) tissue-specific bioinks. The 3D bioprinting process usually comprises the following four steps: (1) scanning the body organs/tissue area using X-rays, computed tomography (CT) scans, or ultrasound scanners; (2) importing the medical images into editable 3D files; (3) importing the medical models and slicing to form printable files (STL files); (4) layer-by-layer printing with tissue-specific bioinks using organ-specific cells [253,254]. Bioink is generally referred to as a biocompatible hydrogel ink mixed with cells for 3D printing [255]. Owing to their attractive biomimicking properties and excellent biocompatibility, naturally derived biopolymers are among the most suitable components for bioink fabrication. Naturally derived hydrogels provide better ECM-mimicking functions than chemically synthesized hydrogels, thus facilitating greater nutrient and oxygen diffusion to printed cells [[255], [256], [257], [258]]. Previous studies have shown that bioprinted constructs may sustain cell viability of up to 90%, depending on the bioink composition and nature of the cell [[259], [260], [261], [262], [263], [264], [265], [266], [267]].

Various bioprinted constructs, such as in vitro organ models with single or multiple cells, have been developed for testing patient-specific tumors, diseases, drug screening, and preclinical therapy to recapitulate the native tissue/organ [[268], [269], [270], [271]]. In the case of precision bioprinting, patient-specific (personalized) cells are isolated from biopsies (autologous donor) to manipulate the 3D microenvironment or non-invisibly print onto the patient's body (also known as non-contact bioprinting). Therefore, patient-specific in situ bioprinting is an attractive tool to fulfill the doctor's need at an operation table with minimal surgery and lower chances of immune rejection [32,272,273]. Besides the 3D bioprinting approach, four-dimensional (4D) printing is a next-generation advanced tool that integrates the spatial and temporal transformation of 3D printed constructs. Recent studies have demonstrated that 4D printing is applicable in the industry and biomedical engineering to develop neural, cardiac, renal, and osteochondral models [[274], [275], [276]]. 4D bioprinting allows additional bioprinting on the 4th axis, which is impossible to obtain in conventional 3D bioprinting. Thus, 3D/4D bioprinting holds great promise for future stem cell-based regenerative medicine by biomimicking live tissues/organs to combat various diseases. In the following two subsections, we discuss the recent progress in 3D bioprinting and immunoengineering for bone tissue regeneration.

8. 3D printing/bioprinting and immunoengineering for bone regeneration

Bone is a highly mineralized tissue that provides mechanical support and protection to internal organs during locomotion [277]. According to a World Health Organization report, the increasing rate of osteoporotic patients worldwide is estimated to be 8.9 million by 2030. Osteoporosis at a later stage becomes fatal, and its postoperative complications are huge [[278], [279], [280]]. Bone marrow transplantation [281] and stem cell therapy [282,283] are two leading strategies for treating osteoporosis. However, this procedure is complicated and may lead to severe threats to life. Therefore, conventional treatment strategies, such as autografts or xenografts, are usually employed to treat osteoporosis. In most cases, graft transplantation may require thousands of dollars, creating a massive socio-economic burden [[284], [285], [286]]. In this regard, 3D bioprinting can overcome the existing challenges in bone tissue engineering, particularly in bone-related diseases [287,288]. 3D bioprinting helps re-create bone-mimicking structures through layer-by-layer printing of bone stem cells with bone-specific bioinks [289]. For example, 3D-printed bioactive glass/β-TCP has been shown to promote bone regeneration owing to its unique microporosity and mechanical stiffness after calcination at high temperatures [290]. However, calcium phosphate (CaP)-based bioceramics have poor bioprintablity owing to their low cell-loading capacity. To address this issue, Chen et al. reported a bioprinting strategy using Li²⁺/Ca²⁺-reinforced silicate nanocomposites to control cell alignment and induce osteogenesis both in vitro and in vivo [291]. Hard and calcified 3D printing is usually achieved through FDM printing [[292], [293], [294], [295]], wherein the 3D printed constructs require post-processing, such as high-temperature sintering or cold isotactic pressing (CIP). Thus, bioprinting is unsuitable for FDM-type 3D printing. Although several attempts have been made to create large-scale bone constructs, the clinical application of printed constructs is still under consideration owing to the availability of suitable bone bioceramics with less immunotoxicity [[296], [297], [298]]. Currently, various biomaterials, such as ceramics, metals, natural and chemical polymers, and polyesters, are used to fabricate bone constructs [[299], [300], [301], [302]]. An ideal bone graft must have desirable mechanical properties, including superior biocompatibility, controlled biodegradability, unique porosity, and immunomodulatory properties. Rapid angiogenesis and macrophage polarization (anti-inflammatory macrophages) during bone injury are other important phenomena in vascularized bone regeneration [303] (Fig. 9a). It has been shown that early activation of VEGF during osteoblast maturation is critical for angiogenesis. MSCs cross-talk with endothelial cells during bone remodeling (Fig. 9b); thus, vascularized bone regeneration in a clinical setting remains a challenge in regenerative medicine [304]. Sun et al. reported that MSN-loaded GelMA/Gelatin/PEG-based 3D bioprinted hydrogel induced the sustained release of BMP-2, which induced M2 polarization of RAW 264.7 cells during osteogenic differentiation [305]. This study also highlighted that the MSN/BMP-2 nanocomposite mainly stimulated anti-inflammatory factors (IL-4 and IL-10), which helped in robust bone regeneration in a calvaria defect model under diabetic conditions (Fig. 9c). In another study, 3D printed monetite (a modified calcium phosphate compound, CaHPO₄)-coated Ti₆Al₄V composite was shown to induce osteoimmunomodulation via the activation of M2 macrophages [306]. Owing to the superior mechanical properties of Ti₆Al₄V (Young's modulus 4–8 times higher than that of human bone) and stiff matrix, the macrophages could differentiate into an anti-inflammatory phenotype and secrete IL-4, which accelerated the osteogenesis of BMSCs (Fig. 9d–f). Table 7 lists various types of 3D bioprinted scaffolds with osteoimmunomodulatory properties for bone tissue engineering applications.

Fig. 9.

(a) Schematic diagram showing the blood vessel organization of an adult bone marrow. Bone marrow comprises numerous H-type blood vessels in the metaphysis region with osteoprogenitor cells. The osteoprogenitor cells support the angiogenesis of the H-type vessel during bone remodeling [303]. (b) Schematic illustration of fracture healing in the femur, which requires a series of molecular cross-talk between bone cells, endothelial cells, and immune cells [307]. (c) Demonstration of 3D bioprinting of MSN-BMP-2 loaded GelMA/gelatin/PEG hydrogels with enhanced osteoimmunity and robust in vivo bone regeneration [305]. (d–f) 3D printed Ti6Al4V scaffolds promote the osteogenic differentiation of BMSCs and enhance vascularized bone regeneration via immunomodulation [306].

Table 7.

Recent advances in 3D printed/bioprinted osteoimmunomodulatory hydrogel scaffolds for bone tissue engineering.

Composition	Monocyte/macrophage polarization	Immunomodulatory activity	References
Glycopeptide-conjugated PCL/nHAp scaffolds	M2 phenotype	β-sheets of Glycopeptide promoted higher expression of STAT-6, IL-10, and TGF-β through ERK signaling pathway which induced the osteoblast differentiation	[228]
Strontium-doped nHAp/silk scaffold	M2 phenotype	Strontium induced the HIF-1α activity and promoted angiogenesis and chondrogenesis; the composite scaffold also boosted the secretion of anti-inflammatory factors and accelerated osteogenesis	[308]
Haversian canal mimicking bioprinting using Ca2MgSi2O7 + BMSCs (2 × 104) + RAW 264.7 cells (2 × 104)	M2 phenotype	The co-culture of BMSC/RAW 264.7 increased the expression of Arg-1, IL-10, and CD163 in RAW 264.7 cells and induced the expression of BMP-2, TGF-β1, PDGF, and VEGF; enhanced osteogenic differentiation through activation of BMP2R/Smad4 signaling	[309]
3D printed β-TCP/Alginate/hyaluronic acid scaffold	M2 phenotype	The soft hydrogel promoted M2 polarization via enhancing the expression of Arg-1, IL-1ra, IL-10, TGF-β1, and VEGFA; the M2 macrophage derived exosome promoted bone regeneration through activation of Runx2, ALP, BSP, BMP-2, OCN, and OPN transcription factors	[305]
3D printed PLA/MSC-exosome	M2 phenotype	3D printed MSC-exosome loaded scaffold promoted M2 macrophage polarization and hBMSCs differentiation via immunomodulation; the MSC-exosome downregulated the pro-inflammatory factors, such as IL-1β, IL-6, iNOS, and TNF-α	[310]
3D bioprinted Dox-loaded MBG/GelMA/HAMA scaffold	M1 phenotype	Enhanced activation of pro-inflammatory cytokines and anti-bacterial effect; sustained BMP-2 release boosted in vivo bone regeneration	[311]
3D printed hydroxypropyl chitin/PLA/nHAp	M2 phenotype	Enhanced activation of M2 macrophages via activation of IL-10, Arg-1, CCL22, VEGFFA, PDGFB, and MMP9; robust in vivo bone regeneration in calvaria defect model through osteo-immunomodulation	[312]
3D/4D printing of polydopamine (PDA)-modified collagen/PGS/PLA scaffold	M2 phenotype	PDA modification induced the M2 polarization of RAW 264.7 cells and sustained the release of anti-inflammatory cytokines for robust craniofacial regeneration	[313]
3D bioprinted AgGNRs/dextran/GelMA scaffold	M2 phenotype	Activation of anti-inflammatory phenotype of RAW 264.7 cells; sustained the secretion of IL-4; enhanced osteoimmunomodulation of BMSCs; anti-bactericidal effect	[314]
3D printed Ca7Si2P2O16 scaffold	M2 phenotype	The multicellular patterning of MSCs and RAW 264.7 cells exhibited a neighborhood effect; enhancement of M2 markers (CD206, Arg-1, IL-10, and IL-1ra); M2 macrophage triggered the osteogenesis via Smad/β-catenin/LRP5 signaling axis and promoted robust bone regeneration in vivo; enhancement of in vivo activation of IL-10 marker	[315]
QCS/GO/PDA-based 3D printed scaffold	M2 phenotype	The nanohybrid scaffold promote M2 macrophage polarization and accelerated bone and skin regeneration	[316]
Cryogenic 3D printing of Sr2+/Fe3+ co-substituted nHAp	M2 phenotype	The metal ions facilitated angiogenesis and osteogenesis through M2 macrophage polarization; secretion of anti-inflammatory factors (IL-10 and Arginase)	[317]

Peptide-based hydrogels have been shown to promote macrophage polarization by inducing the adhesion, elongation, and activation of various signaling pathways. Previous studies have demonstrated that bioactive peptides or peptide-coated metal nanoparticles promote M2 macrophage polarization and elicit protection against xenobiotic-induced organ injury [[318], [319], [320], [321]]. Wang et al. reported that 3D printed glycopeptide (RADA-16)-conjugated PCL/nano-HAp scaffold promoted bone regeneration by activating M2 macrophages in vivo [228]. This study also highlighted that the RADA-16 peptide induced the activation of STAT6/ERK signaling in RAW 264.7 cells and boosted the secretion of anti-inflammatory cytokines, which accelerated the osteogenesis of BMSCs (Fig. 10a and b). Furthermore, it was also demonstrated that RADA-16 coated PCL/nano-HAp scaffold induced higher mineralization and new bone formation (∼88.3%) than the blank scaffold. In another study, MSC/RAW 264.7 cells co-cultured in a 3D printed Ca₂MgSi₂O₇ scaffold exhibited enhanced osteoimmunomodulation during bone regeneration [309]. The RAW 264.7 cell-secreted cytokines (bioactive proteins) promoted the proliferation and differentiation of BMSCs (Fig. 10c and d). Thus, the use of various bioactive protein/peptide coatings may help regulate macrophage activity, which is crucial for bone regeneration. Macrophage-derived secreted biomaterials, such as soluble factors, cytokines, and exosomes, can be used to prepare soft hydrogels for controllable delivery of growth factors/exosomes during osteogenesis. Sun et al. showed that β-TCP induced M2 macrophage polarization, and the secreted exosomes also displayed enhanced angiogenic and osteogenic activity in human umbilical vein endothelial cells (HUVECs) and BMSCs [322]. The M2-exosome of RAW 264.7 cells, when encapsulated in an alginate/hyaluronic acid hydrogel, exhibits controlled release after incubation in culture media and accelerates osteogenesis (Fig. 10e). The authors also demonstrated that pro-angiogenic and pro-osteogenic microRNAs (miRNAs) present in M2 macrophage-derived exosomes play a crucial role in osteo-angiogenesis. Thus, the immune response can be manipulated by controlling scaffold morphology, surface topography, chemical composition, or bioactive protein/peptide coating; this could be beneficial in developing new-generation immunomodulatory scaffolds for bone tissue regeneration.

Fig. 10.

Recent advances in 3D bioprinted osteoimmunomodulatory platforms for robust bone regeneration. (a, b) Glycopeptide-modified polycaprolactone/nano-hydroxyapatite (PCL/nHAp) scaffold (1, 2) promoted robust bone regeneration (3–7) through M2 macrophage polarization (b, 1–5). Scale bars: 50, 200, 500 μm, 1 mm, and 5 mm [228]. (c, d) Demonstration of 3D bioprinted Haversian canal mimicking hydrogel (1–4) for immunomodulation of MSCs and RAW 264.7 cells (5, 6). The fabricated hydrogel promoted anti-inflammatory phenotype activation of RAW 264.7 cells during MSC/RAW 264.7 co-culture. Scale bars: 50 and 100 μm [309]. (e) Fabrication of macrophage exosome-laden alginate hydrogel (1–5) for immunity boosting (6) and enhancing the osteogenic differentiation. Scale bars: 2 and 3 mm [322].

9. Limitations of 3D bioprinting in immunoengineering

3D bioprinting is a groundbreaking AM technology that enables the manipulation of various types of cells with superior complexity and accuracy using a single bioink. Although 3D bioprinting has gained enormous attention in regenerative medicine, many challenges remain unsolved in developing immunomodulatory platforms. One of the significant challenges is the viability and proliferation of immune cells inside the bioprinted hydrogel. The bioink viscosity and modulus (e.g., elastic modulus) play a crucial role in the survival and proliferation of immune cells. Soft hydrogel matrix may promote greater mobility and differentiation of immune cells; however, the stiff hydrogel will not promote cell mobility due to the less availability of nutrients and poor oxygen diffusion. Thus, most of the literature research relies on a simple 2D culture of immune cells onto the 3D printed hydrogels. Therefore, tissue-specific and soft hydrogel matrix development is desirable for enhancing macrophage/monocyte encapsulation efficiency with greater viability. Various decellularized extracellular matrix (d-ECM) bioinks have recently been proposed to tune macrophage polarization during tissue regeneration. However, using d-ECM-based hydrogels for in vivo applications is quite questionable. This is because the cells of the native tissue contain various surface markers and intracellular proteins, which act as a recognition site for several immunocytes. Removal of cellular components by detergents (e.g., sodium dodecyl sulfate and Triton-X 100) more likely remove the surface markers and intracellular proteins, thereby reducing the chance of macrophage/monocyte cells to activate. Thus, the use of only d-ECM hydrogel is quite ineffective for immunomodulation. Therefore, future bioink development must focus on studying not only the d-ECM hydrogels for bioprinting but also focus on immunomodulatory effects for precision medicine. Most fabricated cell-printing platforms demonstrate excellent physiochemical properties towards macrophage culture in vitro; however, they fail to recapitulate the native immune environment when implanted in vivo. For example, the bioprinted structure and their implantation, analysis of immunological markers, FBR, long-term in vivo trackability, and degradation behavior must be addressed before entering human clinical trials.

10. Summary and outlook

In this review, we attempt to document the present status of the immunomodulatory biomaterials and their properties for tissue healing and regeneration. As discussed above, the phenotypic plasticity of monocyte/macrophage profoundly affected by the material property, surface topography, and porosity during interaction with biomaterials [28]. Conventional hydrogels and nanomaterials are insufficient for inducing dynamic macrophage polarization due to the lack of a suitable 3D environment, ECM function, and long-term dynamic stability. To some extent, the hydrogel scaffolds can only initiate the local immune response rather than systemic immunity. As a result, biomaterials' degradation product sometimes induces immunomodulation (adaptive immunity) and is later excised from the body via urine [[323], [324], [325]]. Similarly, nanoparticle-based immunotherapeutic have similar issues with long-term dynamic immunomodulation. Thus, a 3D hydrogel platform that can effectively encapsulate the immune cells and offers greater immunity is highly desirable for successful clinical application. 3D printing is a groundbreaking additive manufacturing tool offering a wide range of biomaterials for rapid prototyping [326]. Although 3D bioprinting and immunomodulation are the ‘hot topic’ in tissue engineering and regenerative medicine; before going forward, some key aspects need to be addressed for the successful application of the immunomodulatory scaffolds. Of them, the major issue is the commercialization and mass production with an eco-friendly and economic approach. Moreover, the biomaterial-assisted immunotherapy should be taken into consideration in terms of safety and body's innate immunity. Furthermore, the 3D bioprinted immunomodulatory hydrogels can also be used as a novel platform for delivering vaccines and drugs individually, known as precision medicine [327].

With the advent of modern biotechnology, various genome editing tools has gained significance attention in the therapeutics. In particular, various nucleic acid-based biopolymers and genes are mixed with other polymers for re-engineering the genetic makeup of the cells. Such genome editing tools are commonly known as ‘God's scalpel,’ which is a precise and efficient tool for editing the DNA or RNA of the cells [328]. One of the promising gene editing tools used in immunology and molecular biology is known as clustered, regularly interspaced, short palindromic repeat or CRISPR after being discovered by Ishino et al., 1987 [328,329]. CRISPR is usually associated with other regulatory genes known as the Cas9 gene. Engineering macrophage cells with CRISPR/Cas9 expression plasmid regulated the specific polarization and enhanced the tissue-specific immunity during inflammation or restoration [[330], [331], [332]]. Even though, commercially available viral or non-viral (PEG-based polymeric nanocarriers) are extensively used for delivery of CRISPR/Cas9 plasmids; however, the release of those nanocarriers from a 3D printed hydrogel is quite questionable. Several factors, such as polymer ink composition, concentration, crosslinking degree, and viscosity is related to the sustained release of various drugs, nanoparticles, and nanocarriers. Therefore, development of novel biomaterial-based nanocarriers with controlled release of CERSPR/Cas9 is highly required for dynamic macrophage immunomodulation (Fig. 11). In addition, extensive in vitro and in vivo analysis is also necessary for the successful clinical application of the CRISPR/Cas9-based 3D hydrogels. Thus, future research also focuses not only developing novel platforms but also integrating CRISPR-Cas9 based advanced tools which can cut, edit, and re-engineer the macrophages towards tissue healing and regeneration.

Fig. 11.

Schematic illustration showing the role of CRISPRized technology with 3D printing/bioprinting technology to regulate macrophage polarization.

Macrophages interact with stem cells to secrete various growth factors and cytokines which may induce the proliferation and differentiation of other cells. In addition to biomaterials properties, including stiffness/elasticity, topography, and porosity, various biophysical simulations have also been shown to modulate cell adhesion, proliferation, and differentiation [[333], [334], [335], [336]]. For instance, electric field (EF) [246,337,338], magnetic field (MF) [339], hydrostatic pressure [340], and electromagnetic field (EMF) [341] can also be used to stimulate biomaterials for dynamic immunomodulation and regeneration. Various electro active polymers (EAPs) and magneto-active polymers (MAPs) or their composites have been extensively studied in the past few years for understanding mechanobiological cues of cell adhesion and differentiation [[342], [343], [344]]. In this context, the development of novel and stimuli-responsive materials should be required for studying the immunomodulation of macrophages in the future for a better understanding of the effects of biophysical stimulation. Recently, the phenotypic classification of macrophage is migrated towards smart-computer-based identification techniques for better interpretation of immune cells [[345], [346], [347]]. Conventional identification procedures of macrophages include the use of fluorescence (FL) microscopy, quantitative real-time PCR (qRT-PCR), and western blotting (WB), which usually takes a longer time to perform the experiments [[348], [349], [350], [351], [352]]. On the other hand, morphological identification via digital or optical microscopy is relatively simple and effective and provides a gross overview of macrophage polarization based on surface morphology. Among various computer-based morphometric tools, artificial intelligence (AI), machine learning (ML), single-cell autofluorescence (SCF), and Raman spectroscopy (e.g., surface enhanced raman spectroscopy or SARS) tools are ‘hot spots’ in biological research owing to the high-accuracy (∼90%) of prediction and cost-effectiveness. Next-generation multi-stimulation-assisted bioprinting platforms must integrate biochemical and biophysical properties that can dynamically modulate macrophage polarization during bone healing and regeneration.

Ethics approval and consent to participate

This review article does not require any ethical approval or allied consents for publication

Declaration of competing interest

The authors declare no competing financial interests related to this article.

Appendix A. Supplementary data

The following are the Supplementary data to this article.

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