Immunomodulatory Biomaterials and Emerging Analytical Techniques for Probing the Immune Micro-Environment

Abstract

After implantation of a biomaterial, both the host immune system and properties of the material determine the local immune response. Through triggering or modulating the local immune response, materials can be designed towards a desired direction of promoting tissue repair or regeneration. High-throughput sequencing technologies such as single-cell RNA sequencing (scRNA-seq) emerging as a powerful tool for dissecting the immune micro-environment around biomaterials, have not been fully utilized in the field of soft tissue regeneration. In this review, we first discussed the procedures of foreign body reaction in brief. Then, we summarized the influences that physical and chemical modulation of biomaterials have on cell behaviors in the micro-environment. Finally, we discussed the application of scRNA-seq in probing the scaffold immune micro-environment and provided some reference to designing immunomodulatory biomaterials. The foreign body response consists of a series of biological reactions. Immunomodulatory materials regulate immune cell activation and polarization, mediate divergent local immune micro-environments and possess different tissue engineering functions. The manipulation of physical and chemical properties of scaffolds can modulate local immune responses, resulting in different outcomes of fibrosis or tissue regeneration. With the advancement of technology, emerging techniques such as scRNA-seq provide an unprecedented understanding of immune cell heterogeneity and plasticity in a scaffold-induced immune micro-environment at high resolution. The in-depth understanding of the interaction between scaffolds and the host immune system helps to provide clues for the design of biomaterials to optimize regeneration and promote a pro-regenerative local immune micro-environment.

Graphical abstract

Introduction

The host immune responses following biomaterial implantation are collectively called the foreign body response (FBR) [1]. This sequential reaction includes protein adsorption on the scaffold surface, inflammatory cell recruitment and infiltration, foreign body giant cells (FBGCs) formation, activation of fibroblast, and ultimately fibrotic encapsulation [2]. Scaffolds implanted can be made of natural or synthetic materials, in the forms of films, nanofibers, hydrogels, et al. [3]. When implanted in vivo, synthetic materials showed high immunogenicity and led to severe fibrotic response. The extracellular matrix (ECM) scaffold treated by decellularization treatment has well solved the problems above and showed good biocompatibility in native tissue [4, 5]. Since recruitment of immune cells is dictated by the tissue location where the biomaterial is implanted, degree of trauma, and the physiochemical properties of the scaffold [6], efforts have been made to improve the surgical procedures and scaffold design [7–9]. Different biological uses of the material require different physical and chemical properties, in some occasions, scaffolds are expected to function with very low degeneration and minimal fibrotic encapsulation [10], whereas others are designed to keep pace with tissue growth [11]. Biomaterials are therefore envisioned to be capable of modulating the local immune micro-environment to induce a favorable condition for tissue regeneration [3, 12]. The strategies of scaffold design mainly focus on the adjustment of physical cues and surface chemistries of materials [13]. The physical cues for modulation mostly including fiber size, stiffness, wettability, surface topography, pore size, etc., affect the adsorption of proteins and the biological reaction of immune cells to influence the process of acute and chronic inflammatory reactions, resulting in different outcomes of tissue fibrosis or regeneration [14–17]. Chemical methods include material composition, surface chemical modification, combination of bioactive substances. By reducing the first step of protein adsorption, then alleviating successive inflammatory responses, or releasing bioactive molecules with immunomodulatory function, the local immune micro-environment can be mediated into the direction of promoting tissue regeneration [14].

Immune cells such as granulocytes (neutrophils, eosinophils, basophils), mast cells, macrophages, dendritic cells, and lymphocytes (B cells, T cells) play different roles in mediating the foreign body response [18]. Neutrophil infiltration is predominant for the initial two days following implant placement. Then at day 3, the primary immune cell type becomes tissue-resident macrophages or monocyte-derived macrophages [19, 20]. Cells of the monocyte-macrophage lineage are highly heterogenous and plastic [21]. Macrophages might transition into pro-inflammatory M1 macrophages [induced by toll-like receptor (TLR) ligands and interferon-γ, (IFN-γ)] or anti-inflammatory M2 activation [induced by interleukin-4/interleukin-13, (IL-4/IL-13)] dictated by diverse environmental cues. The polarization of macrophages mirrors the T helper 1 (Th1)-Th2 transition of T cells [22, 23]. Recently, numerous studies have focused on the regulation of M2 macrophages on tissue regeneration and further explored the specific phenotype controlling regeneration and how these phenotypes switch within this heterogeneous and versatile lineage [24, 25]. Other cells besides macrophages also determine whether the tissue is headed for fibrosis or regeneration. The phenotypes, activation states and differentiation trajectories of immune cells in different anatomical niches have not been fully understood. In the past decade, emerging technologies like scRNA-seq and mass cytometry allow more accurate and high-resolution detection of the biosystem [26, 27]. Our understanding of the heterogeneity of immune cells populations, functions, and the diversity of their phenotypes continues to deepen [28, 29].

In this review, we briefly discuss the processes of foreign body response (FBR) and different types of immune response after biomaterial implantation. Then, we summarize the physical and chemical strategies used in developing immunomodulatory biomaterials. Finally, we discuss the application of scRNA-seq in probing the scaffold immune micro-environment to better inform biomaterials-directed regenerative immunoengineering design.

FBR and immune micro-environment around biomaterials

Foreign body response

Implantation of biomaterials can cause a series of chain reactions including protein adsorption, acute and chronic inflammatory reactions, formation of foreign body giant cells and fibrosis, which are collectively called FBR [7]. After implantation of the scaffold, proteins from blood and other body fluids, including albumin, fibronectin, fibrinogen, and chemoattractants generated by activated complement system, adhere to the biomaterial surface by intrinsic contact activation of the coagulation cascade and the alternative pathway of activation of completement system, forming provisional matrix, which is determined by biomechanical properties of materials [30, 31]. Then inflammatory cells possessing adhesion receptors interact with the adsorbed proteins on scaffold surface by pathogen-associated molecular pattern (PAMPS) and integrin-mediated interaction, leading to recognition of the biomaterials [1].

During the second stage of FBR, namely acute inflammation, granulocytes and mast cells infiltrate into the area. The damage associated molecular patterns (DAMP) like heat-shock proteins, and double-stranded DNA are released, and recruit granulocytes to the implantation sites [30, 32]. Neutrophils engulf and destroy cell debris by antibody-dependent cell-mediated cytotoxicity (ADCC) [33]. These inflammatory cells then produce cytokines like IL-8, MCP-1, IL-4, IL-13, MIP1b, histamine and CXCL13. The released cytokines and chemokines recruit more leukocytes, especially macrophages, to the wound defect area to establish the chronic inflammation phase [34, 35]. Driven by DAMPs and natural killer cell-derived interferon-γ, macrophages polarize towards pro-inflammatory phenotype (M1) [36]. M1 macrophages synthesize matrix metalloprotease (MMPs) that can digest ECM and aid in their migration [32, 37, 38].

With the resolution of acute inflammation, the M1 macrophages polarize towards the alternatively activated M2 macrophage [13, 36]. M2 macrophages are further divided into M2a, M2b, M2c, and other subtypes, among which M2a and M2c subtypes promote angiogenesis and tissue repair [39–41]. During wound healing, colony stimulating factor-1(CSF-1)–dependent macrophages promote neoangiogenesis by secreting MMPs to accelerate matrix remodeling [42].

Macrophages fuse into FBGCs, which is considered a hallmark of FBR [34], perhaps to improve their functions or to avoid apoptosis [43]. This phenomenon appears to be harmful to biocompatibility of the scaffolds, and is regarded as a target for intervention [44]. As the chronic inflammation and FBGC formation proceed, a collagenous envelope surrounding the scaffold builds up. Triggered by pro-fibrotic signals, like TGF-β, fibroblasts and endothelial cells deposit collagen and other extracellular proteins on the surface of scaffolds [30, 34, 45]. This layer of granulation tissue may grow into a thick fibrotic tissue with extremely few infiltrated immune cells and poor vascularization, which might eventually lead to local infections [46]. The immune cells, inflammatory cytokines, chemokines, and receptors involved in FBR are summarized in Table 1.

Table 1.

Cells, proteins, cytokines and chemokines involved in FBR

No.	Phase of FBR	Cell type	Cytokine	Chemokine	Protein	Refs.
1	Protein adsorption				Albumin, fibrinogen, kninogen, fibronectin, vitronectin, C3a, C5a	[1, 30, 31]
2	Acute inflammation	Granulocytes, mast cells, neutrophils, macrophages, endothelial cells, natural killer cells	IL-1,IL-4, IL-6, IL-8, IL-12, IL-13, MCP-1, MIP1b, histamine, interferon-γ,TNFα	Coagulation factor VII, XI, von Willebrand factor (vWF), PF4 or P-selectin,CXCL13	DAMPs, MMPs, integrin receptors, Toll-like receptor (TLR)	[30, 32–38]
3	Chronic inflammation	Macrophage/monocytes, lymphocytes, mast cells	CSF-1, TGF-β, PDGF, MIP-1a, MIP-1b, IL-6, IL-8, TNF-α, IL-13, IL-4, IL-10	Complement factors, PF4, MCP-1,2,3,4, RANTES	Mmps, TLR, scavenger receptors, integrin receptors	[13, 36, 39–42]
4	Formation of foreign body gigantic cells	Foreign body giant cells (FBGC), T cells, mast cells	IL-13, IL-4, IL-1a; IL-6, IL-8, TNF-α, IL-10, TGF-β	MCP-1	β-integrin receptors, mannose receptors, CD44, CD47, E-cadherin, CD11, CD45	[34, 43, 44]
5	Fibrosis	FBGC, endothelial cells, fibroblasts, myofibroblasts	TGF-β, PDGF, VEGF		MMPs	[30, 34, 45, 46]

Immune micro-environment around biomaterials

Scaffold implanted, local tissue environment and immune cells together constitute the local immune micro-environment [47]. Immune cells (neutrophils, macrophages, monocytes, NK cells, lymphocytes, mast cells, etc.) are the first fighters confronted with foreign bodies, and then send signals to activate fibroblasts in the tissue environment. The intercellular and intracellular signaling networks between immune cells and stromal cells lead to different physiological outcomes by directing cell behaviors and secreting cytokines, chemokines, or active substances [47].

In the regeneration process regulated by immunomodulatory biomaterials, cells of the innate and adaptive immune system play different, yet interactive roles. The type 1 immune polarization, driven by Th1(T-helper cell 1) cells from the adaptive immune system, is considered pro-inflammatory. M1 macrophages express nitric oxide synthase (iNOS) and tumour necrosis factor (TNF) in response to the stimulation of IFN-γ produced by Th1 cells, TLRs, and other intracellular pattern recognition receptors. In reverse, cytokines and chemokines secreted by M1 macrophages, e.g., IL-12, CXCL9, and CXCL10, can recruit Th1 cells and promote the Type 1 polarization [48]. On the contrary, Type 2 immune polarization is characterized by the production of type 2 cytokines such as IL-4, IL-5, IL-9, IL-13, and is related to tissue repair and regeneration after wounding. Main cell types associated with type 2 immunity include Th2(T-helper cell 2) cells, macrophages activated by type 2 cytokines, mast cells and type 2 innate lymphoid cells [6, 49, 50]. Type 17 immune response has also been reported to promote chronic fibrosis in tissue around breast implants. Implantation of synthetic materials increased the expression level of IL-17 secreted by group 3 innate lymphoid cells, γδ T cells, and CD4⁺ adaptive T cells (Th17). It has been shown that elevated IL-17 expression was associated with fibrosis, while the blocking of IL-17 signaling reduced fibrotic response [51].

Synthetic and biological materials usually induce diverging immune micro-environments. When used in muscle wound, uncrosslinked ECM scaffolds induced increased levels of Th2-associated genes and genes related to DAMP signaling (Mbl2, Clec7a), which might contribute to the formation of pro-regenerative immune environment [52]. On the contrary, synthetic materials were usually associated with extensive and chronic neutrophil infiltration and various extents of fibrosis, depending on the material’s stiffness and size [52]. The exploration of immune cell activities around specific biomaterials implanted will provide new clues for the design of immunomodulatory materials.

Advances in the design and fabrication of immunomodulatory biomaterials

Upon implantation of biomaterials, host immune response might lead to impaired device function [53, 54]. To engineer immunomodulatory biomaterials, researchers usually focus on the incorporation and controlled release of immunomodulatory agents, regulation of target cell population, minimizing systemic toxicity, and modulation of scaffold physicochemical properties [55–59]. Here, we summarize the features of some of the immunomodulatory biomaterials used in soft tissue environment (skin/muscle). (Table 2).

Table 2.

Physical and chemical modifications and the modulatory effects of biomaterials

No.	Author	Modification methods	Target immune cell	Modulatory effects	References
Physical modification
1	McWhorter et al.	Micropattern	Macrophages	Elongation of macrophages stimulated polarization toward an M2 phenotype	[60]
2	Luu et al.	Micro and nano-pattern	Macrophages	Microscale grooves activated M2 phenotype	[57]
3	Friedemann et al.	Matrix stiffness	Macrophages	Increase in matrix stiffness led to an anti-inflammatory macrophage phenotype	[61]
4	Abebayehu et al.	Fiber size and pore diameter	Mast cells	Pore size ≥ 4 μm diminished inflammatory cytokine production	[62]
5	Veiseh et al.	Dimensions of spheres	Macrophages	Spheres of ≥ φ1.5 mm mitigated foreign reactions and fibrosis	[56]
Modulation of surface chemistries
6	Swartzlander et al.	Oligopeptide RGD	Macrophages	RGD reduced the fibrous capsule density and thickness	[63]
7	Alapure et al.	Mesenchymal stem cells loaded	Macrophages	Increase in M2 macrophages, and reduction of M1 macrophages	[64]
8	Zhang et al.	Prostaglandin E2	Macrophages	Promoting the M2 phenotypic transformation of macrophages	[55]
9	Sok MCP et al.	AT-RvD1 and IL-10	Mononuclear phagocytes	Recruiting CD206+ macrophages (M2a/c) and IL-10 expressing dendritic cells	[65]
10	Yan et al.	Sialidase	THP-1, macrophages	Transiently activating macrophages in a sialic acid-dependent manner	[66]
11	Dong et al.	IFN-γ, mesenchymal stem cells loaded	Macrophages	Promoting polarization of M2 macrophages, reducing FBR, and avoiding tendon adhesion	[67]
12	He et al.	Modifying L-nitroarginine and PEA copolymers	Macrophages	Reducing NO, and increasing the arginase activity in macrophages	[68]
13	Jiang et al.	Mannose-decorated globular lysine dendrimers (MGLDs)	Macrophages	Targeting M2 polarization, inhibiting the secretion of pro-inflammatory cytokines and increasing the production of TGF-β1	[69]
14	Welch et al.	B7-33	Fibroblasts	Reducing the fibrous encapsulation	[70]
15	Chu et al	EGCG	Macrophages	Promoting M2 polarization	[71–78]

Modulation of physical cues

Microscale and nanoscale structural properties of biomaterials can regulate cellular responses and alter cell fates [79]. In the internal environment, physical cues of the scaffolds, including fiber size, stiffness, wettability, surface topography and porosity of the biomaterial, can modulate FBR by tuning phenotypes of immune cells [56, 58, 80]. Nanofiber structure is more similar to extracellular interstitial structure according to its porous structure and high specific surface area, which facilitates cell adhesion, nutrient exchange and waste transport, and can guide specific immune cell phenotypic differentiation and promote tissue maturation [79, 81, 82]. Dhivya et al. summarized matrix characteristics that promote anti-inflammatory immunophenotypes by concluding the regulation of specific fiber diameter, aperture, and fiber orientation on the fate of innate immune cells [83]. In response to diverse surface topography of ECM structures, bone marrow-derived macrophages showed different cell shape. According to Liu et al., cell elongation enhanced the expression of M2-related arginase-1 and mitigated the expression of M1-related iNOS [60]. The same group also found that cultured on groove sizes ranging from 400 nm–5 μm in width are along with higher levels of anti-inflammatory IL-10 secrection of macrophages [57]. As for engineered 3D Coll based matrices, variation of matrix stiffness by EDC crosslinking and covalent glycosaminoglycan (GAG) modification affected the polarization states of macrophages [61]. An increase in matrix stiffness resulted in an anti-inflammatory macrophage phenotype [61]. Mast cells were also necessary in FBR [84]. When they were seeded on electrospun polydioxanone (PDO) scaffolds with diverse fiber size and pore diameter and stimulated with IL-33 or lipopolysaccharide (LPS), inflammatory cytokine production was greatly reduced on scaffolds with larger pore size (at or more than 4 μm) [62]. To explore the role of spherical biomaterial geometry on FBR, Zandstra et al. injected monodisperse microspheres of defined size and polydisperse microspheres under rat skin. Macrophage infiltration and collagen encapsulation increased in small microspheres compared to large ones [85]. It was further demonstrated that the spherical shape and spheres with diameter of 1.5 mm or greater significantly alleviated foreign reactions and fibrosis [56].

Modulation of surface chemistries

Molecules, drugs, and ions are often applied to biomaterials to regulate FBR [70]. Among the candidate materials, hydrogels with the properties of high permeability, low immunogenicity and adjustable mechanical properties can mimic physicochemical structure of natural ECM to restore and rebuild tissue function and were used to treat soft tissue wounds in a number of studies [86, 87]. To reduce protein adsorption at the first step of FBR, some authors incorporated arginine-glycine-aspartic acid (RGD) into poly (ethylene) glycol (PEG) hydrogels, which reduced the density and thickness of fibrotic capsules [63]. Alapure et al. synthesized a biodegradable hybrid hydrogel (ACgels) and evaluated its effects in burn wounds. Significantly higher re-epithelialization and angiogenesis was observed for the ACgels seeded with mesenchymal stem cells [64]. Zhang et al. incorporated PGE2 into chitosan (CS) hydrogel. The composite hydrogel enhanced the secretion of anti-inflammatory cytokines in macrophages and promoted angiogenesis in vitro, characterized by increased expression of M2-associated genes [55]. Sok et al. combined aspirin-triggered resolvin-D1 (AT-RvD1) and IL-10 in the hydrogel synthesis process. AT-RvD1 can mediate the reorientation of immune cell, limit neutrophil migration, and regulate macrophage maturation. The combined application of AT-RvD1 and IL-10 enhanced the expression of macrophages and dendritic cells in the tissue defect area, and promoted wound healing by facilitating the recruitment of anti-inflammatory macrophages [65]. In addition, mucins can also be added into hydrogels as regulatory molecules, transiently activating macrophages possibly by influencing sialic acid receptors such as siglecs [66].

In addition to hydrogels, tissue-derived ECM and other synthetic materials can be chemically modified to regulate surrounding immune micro-environment as well. Dong et al. modified polycaprolactone/silk fibroin (PCL/SF) composite fibrous scaffold with ECM derived from MSC, and stimulated ECM with IFN-γ to obtain immunomodulatory ECM (iECM). They found that iECM-modified scaffold promoted polarization of M2 macrophages to reduce FBR and tendon adhesion [67]. L-nitroarginine (NOArg) based polyester amide (NOArg-PEA) and NOArg-Arg PEA copolymers can be chemically modified to tune levels of NO, cytokine, and growth factors produced from macrophages. The application of NOArg-Arg co-PEA to diabetic rat skin wound promoted wound healing and re-epithelialization, lowered inflammatory cell infiltration, and induced higher M2/M1 ratio [68]. Jiang et al. implanted mannose-decorated globular lysine dendrimers (MGLDs) into full-thickness skin defect of type 2 diabetic mice. This design also effectively promoted the wound healing by targeting M2 polarization, inhibiting pro-inflammatory cytokines secretion and promoting the production of TGF-β1 [69]. B7-33, a truncated B-chain analogue of relaxin (antifibrotic), was incorporated into poly(lactic-co-glycolic) acid (PLGA) coatings. The sustaining release of B7-33 reduced the fibrous encapsulation surrounding a subcutaneous implant [70, 88]. Collagen membranes cross-linked by Epigallocatechin-3-gallate (EGCG) were able to improve cell proliferation and differentiation [71–74], and promoted bone regeneration when combined with nanohydroxyapatite coatings [75]. According to more recent findings, EGCG-modified collagen membranes successfully ameliorated FBR by enhancing M2 polarization of macrophages [76, 77], which might be associated with C–C chemokine receptor type 2 (CCR2) signaling [78].

Current available skin and gingival grafts and future perspectives

The currently available soft tissue grafts in clinic are summarized in Table 3 [89–95]. The skin and gingival substitutes are either biological (autologous, allogeneic, xenogeneic) or synthetic, some of them contain autologous or allogeneic cells. For volumetric muscle loss (VML) injuries, however, clinical treatments besides autografting are lacking. Engineered muscle graft by means of electrospinning, bioprinting, dielectrophoresis, and microfluidic techniques were reported [79, 96, 97]. So far, no commercially available tissue engineered skin grafts possess all properties of an ‘ideal’ skin substitute [92]. The above-mentioned immune-modulatory biomaterials (listed in Table 3) might provide references for the design of future soft tissue grafts.

Table 3.

Current commercially available soft tissue grafts [89–95]

Manufacturer	Description	Indications
Skin: commercially available or marketed dermo-epidermal skin grafts
Allograft
Allograft	Native human skin with cells	Temporary dressing for large burns
Karoskin	Native human cadaver skin
Cellular-autologous
Epicel	Autologous keratinocytes transplanted using petrolatum gauze support	Burns and congenital nevus
EpiDex	Cultured hair follicle keratinocytes (confluent cell sheet)	Chronic leg ulcers
CellSpray	Autologous keratinocytes (subconfluent cell suspension)	Partial-thickness and donor site wounds
MySkin	Cultured autologous keratinocytes seeded on silicone supported layer	Ulcers, superficial burns and skin graft donor sites
PolyActive	Autologous keratinocytes and fibroblasts cultured in PEO/PBT scaffold	Partial-thickness wounds and skin graft donor sites
TissueTech autograft system	Autologous keratinocytes and fibroblasts seeded in a hyaluronic acid scaffold	Diabetic foot ulcers, ischaemic and neuropathic wounds, post-surgical ulcers
Laserskin (Vivoderm)	Autologous keratinocytes seeded on a hyaluronic acid membrane	Excised wounds
Bioseed-S	Autologous keratinocytes (subconfluent cell suspension) on fibrin sealant	Chronic ulcers
Hyalograft	Autologous fibroblasts seeded on hyaluronic acid ester scaffold	Feet ulcer
Acellular
Integra	Dermal layer: bovine collagen-chondroitin-6-sulfate matrix; epidermal layer: synthetic silicone polymer	Deep partial- or full-thickness burns
Biobrane	Semipermeable silicone membrane bonded to nylon fabric	Temporary wound covering for partial-thickness excised burns and donor sites
AlloDerm	Human acellular lyophilized dermis	Dermal graft for burns and other wounds
Terudermis	Fibrillar atelocollagen and heat-denatured collagen	Dermal or mucosal defect
Pelnac	Silicone fortified with silicone gauze TREX, atelocollagen derived from pig tendon	Temporary dermal substitute matrix for all skin loss wounds
SureDerm	Human acellular lyophilized dermis	Hypertrophic scar revision and burns
GraftJacket	Human acellular pre-meshed dermis	Tendon and low extremity wounds repair
Matriderm	Bovine non-cross-linked lyophilized dermis, coated with a-elastin hydrolysate	Deep dermal defects and a split-thickness skin graft
Permacol	Porcine acellular crosslinked dermis	Complex and recurrent hernia repair
OASIS wound matrix	Porcine acellular lyophilized small intestine submucosa	Partial and full-thickness wounds, tunneled wounds, and ulcers
EZ Derm	Porcine aldehyde cross-linked reconstituted dermal collagen	Partial-thickness burns
Hyalomatrix PA	Hyaluronic acid derivatives layered on silicone membrane	Deep partial-thickness burns, wounds after dermabrasion, and deep paediatric burns
Cellular-allogeneic
TransCyte	Neonatal fibroblasts seeded on silicon film, nylon mesh, porcine dermal collagen	Temporary covering for excised deep partial- and full-thickness burns before autografting
Dermagraft	Neonatal fibroblasts seeded on polyglactin mesh scaffold	Full-thickness chronic diabetic foot ulcers
Apligraf	Allogeneic neonatal keratinocytes and fibroblasts cultured in bovine collagen gel	Chronic foot ulcers and venous leg ulcers; burn wounds and epidermolysis bullosa (EB)
OrCel	Allogeneic keratinocytes and fibroblasts cultured in bovine collagen sponge	Split-thickness donor sites
Gingiva: marketed gingival substitute
Mucograft	Porcine collagen matrix without cross-linking	Recession coverage and regeneration of keratinized mucosa around teeth and implants
Fibro-Gide	Porcine, porous, resorbable and volume-stable collagen matrix with smart chemically cross-linking	Thicken the soft tissue around teeth and implants and under pontics
Mucograft Seal	A ready-to-use matrix with a convenient circular shape	Cover extraction sockets during Ridge Preservation

This list might not be all-inclusive

The continuous exploration on the modification of physical and chemical properties of materials has achieved gratifying results. Most of the researches focused on the characterization of material properties and the exploration of superficial biological and immunological reactions, while the in-depth study of the complex and dynamic immune micro-environment around materials is relatively lacking [47]. The deepening interpretation of the interaction mechanism between materials, local tissue and immune cells may provide possible cellular targets for the design of immunomodulatory materials.

Application of single-cell RNA-seq analysis in studying host response against biomaterials

Advances in biological techniques at the protein and gene levels have led to a deeper understanding of the cellular responses surrounding the biomaterials implanted. Traditional protein-level detecting biotechnologies like flow cytometry, western blot, and immunohistochemical/immunofluorescent staining are able to classify immune cells roughly according to few predefined surface markers, but there exists a certain bias. Conventional gene-level techniques such as quantitative polymerase chain reaction (qPCR) can measure population gene expression level based on pre-determined maker genes [98]. However, these markers might be expressed by multiple cell lineages, and plasticity of immune cells may lead to inaccurate classification [99]. In recent years, new analytical techniques have been developed, such as proteomics, mass cytometry, spectral flow cytometry, single-cell transcriptomics, and spatial transcriptomics. These techniques provide powerful tools for in-depth analysis of the immune micro-environment around materials [100, 101].

Bulk RNA-seq can help us understand the molecular mechanisms involved in specific biological process through the analysis of differential gene expression or the entire transcriptome in a mixture of tissues or cell populations [102, 103]. Nevertheless, this type of analysis did not reveal differences between individual cells, which was common in an internal environment regulated by numerous influencing factors [104]. The application of scRNA-seq has enabled thousands of gene expression to be studied at the level of individual cells simultaneously [105, 106], which is being widely applied to discover rare cell types, immunology, tumor heterogeneity, and human disease progress and treatment [107–109]. In recent years, scRNA-seq is increasingly being used to reveal meaningful cell-to-cell gene expression [108, 110, 111], disclose key processes in cell development [112–114], uncover signaling pathways involved in biological processes [115], and reflect intricate intercellular communications in the micro-environment [116].

By using scRNA-seq, previous studies have uncovered unknown heterogeneity of dendritic cells, CD127 + innate lymphoid cells, and pathogenic Th17 cells to name a few [117–120]. In recent years, studies on the biomaterial-induced immune micro-environment at high resolution are emerging (Table 4). Cherry et al. implanted biologic (ECM) and synthetic (PCL) biomaterial scaffolds into murine muscle defects. By analyzing the map of transcriptome, they characterized previously undefined cell subpopulations including NK cells and fibroblast subpopulations [47]. Huang et al. applied high fiber density and low fiber density silk scaffold to rat models. They found previously undefined macrophage subpopulations that are related to the loss of function of the biomaterials implanted: Mmp12⁺/Spp1⁺ (MD1), Mmp9⁺/mk67⁺ (MD2), Mt3⁺/Ckb⁺ (MD3), and further evaluated their functions under different fiber densities [121]. In another investigation, human split-thickness skin grafts (hSTSGs) were implanted into murine full-thickness excisional wounds. Using scRNA-seq, the authors identified a specific subpopulation of macrophages with high expression of the lipid receptor Trem2 (triggering receptor expressed on myeloid cells), which showed a great potential to accelerate wound repair [122]. In the study by Sommerfeld et al., murine volumetric muscle injuries received treatment with ECM or synthetic biomaterial. CD45⁺CD64⁺F4/80^hi+ macrophages were sorted for single-cell analysis. Nine clusters were computationally determined and could be distinguished by Cd301b, Cd9, and Cd74 [123]. Hu et al. dissected the immune micro-environment around electrospun scaffolds implanted into murine skin excisional wounds. Immune cells including macrophages and T cells showed great heterogeneity. They also found that the aligned scaffold fiber structure can accelerate the transition from innate immunity to adaptive immunity [124]. In addition to T cells and macrophages which are mostly explored, the function and heterogeneity of B cells around biomaterials are gradually being explored. To investigate the immune response of B cells to implanted biomaterials, Moore et al. implanted natural biomaterial (ECM) or synthetic biomaterial (PCL) into muscle wounds, and revealed the phenotype and differentiation of B cells using scRNA-seq. The results showed that ECM treatment accelerated germinal centers formation in spleen and lymph nodes. On the contrary, PCL group prolonged the appearance time of B cells and induces B cell antigen presentation and fibrosis [125]. In summary, all these findings mentioned above provide a deeper understanding of immune micro-environment around biomaterials, which may offer potential targets for future immunomodulatory biomaterial design [121].

Table 4.

Studies that analyze the immune micro-environments around scaffolds by using single-cell RNA-seq analysis

No.	Authors	Scaffold implanted	Experimental model	Key findings	Referenecs
1	Cherry et al.	ECM and PCL	Murine volumetric muscle injuries	Characterized previously undefined cell subpopulations including NK cells and fibroblast subpopulations	[47]
2	Huang et al.	High fiber density and low fiber density silk scaffold	Murine skin excisional wounds	Found previously undefined macrophage subpopulations related to the loss of function of the biomaterials implanted: Mmp12+/Spp1+ (MD1), Mmp9+/mk67+ (MD2), Mt3+/Ckb+ (MD3)	[121]
3	Henn et al.	Human split-thickness skin grafts	Murine full-thickness excisional wounds	Identified a specific subpopulation of macrophages with a high expression of the lipid receptor Trem2	[122]
4	Sommerfeld et al.	UBM and PCL	Murine volumetric muscle injuries	The macrophage phenotypes associated with regeneration were defined as R1 (phagocytic F4/80+CD301b+CD9−CD206+) and R2 (non-phagocytic F4/80+CD301b+CD9+CD11c+). CD301b−CD9hi macrophages were related to fibrosis, which were dependent on IL-17 signaling and associated with autoimmunity	[123]
5	Hu et al.	Electrospun scaffolds	Murine skin excisional wounds	Immune cells including macrophages and T cells showed great heterogeneity. Topography of scaffolds controls the T cell response	[124]
6	Moore et al.	ECM and PCL	Murine volumetric muscle injuries	Implanted ECM scaffolds accelerated germinal center formation in spleen and lymph nodes while PCL scaffolds prolonged the appearance time of B cells and induces B cell antigen presentation and fibrosis	[125]

High-throughput sequencing techniques such as bulk tissue RNA sequencing (bulk RNA-seq), scRNA-seq and proteomics can provide massive information on the gene and protein expression profiles of tissue surrounding scaffolds. A brief workflow that can be used in studying scaffold-induced micro-environment using sequencing techniques is shown in Fig. 1 [105, 126]. After in vitro and in vivo evaluation of the biomaterial, harvested samples can be used for bulk RNA-seq, which reveals the differences in gene expression between groups, function of enriched gene sets, signaling pathways for enriched genes, as well as the interplay among them. For scRNA-seq, besides gene level analysis as listed above, cellular analysis including defining cell identity and states, discovering new cell types, and generating pseudotime trajectories can be realized [123, 127]. So far, studies that use scRNA-seq technique to explore host response to biomaterials are in their infancy. Future developments in spatiotemporal profiling of single cells will aid in in-depth understanding of the communication among immune cells, stem cells, and other cell types in regeneration or fibrosis induced by biomaterials [26, 128].

Fig. 1.

A brief workflow that shows how sequencing techniques can be used in studying scaffold-induced micro-environment. After in vitro and in vivo evaluation of the biomaterial, harvested samples can be used for bulk RNA-seq. Through the analysis of PCA or tSNE dimensional-reduction data, gene expression differences between groups, gene functions, signaling pathways and gene network can be interpreted. For scRNA-seq, besides gene level analysis as listed above, we can also analyze from cell level including identifying subpopulations, defining cell states, inferring cell developmental trajectories over the course of a dynamic process and building public databases of important organs. These results may provide better instructions for material design.

Conclusion

The foreign body response consists of a series of biological reactions. The implanted biomaterials, together with immune cells, local tissue environment constitute the local immune micro-environment. The communication between and within cell populations through receptor-ligand interactions and the secretion of molecules and factors forms a signal network, which determines different outcomes of tissue repair or fibrosis. Implantation of synthetic and biological materials usually induce divergent immune micro-environments. By regulating the physical and chemical properties of synthetic and biological scaffolds, materials are designed to provide immune micro-environments that mitigate fibrosis and render the recruited cells polarize towards regenerative phenotypes. In recent years, new technologies like scRNA-seq open a new avenue to identify the rare populations of immune cells that may be activated by biomaterial implants and to examine global transcriptomic changes in different sub-populations of immune cells, which had not been possible with existing technologies. With the help of high-throughput sequencing techniques, massive information on the gene and protein expression profiles of tissue surrounding scaffolds can be obtained to elucidate scaffold-induced micro-environment. Understanding the immune response against biomaterials will aid in the efficient design of biomaterials.

Conflict of interest

There are no conflicts of interest related to this manuscript.

Ethical statement

This article does not contain any studies with human or animal subjects.