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Y. Zhou, P. Yu, J. Xu, R. Luo, C. Zhou, Z. Xiang, P. M. Rommens, M. Liu and U. Ritz, J. Mater. Chem. B,
2023, DOI: 10.1039/D3TB00727H.
Volume 6
Number 3
21 January 2018
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Page 1 of 71 Journal of Materials Chemistry B

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1 Integrated osteoimmunomodulatory strategies based on DOI: 10.1039/D3TB00727H

2 designing scaffold surface properties in bone regeneration

Journal of Materials Chemistry B Accepted Manuscript

3 Zhao Chen1†, Fei Xing1†, Yuxi Zhou2, Peiyun Yu3, Jiawei Xu1, Rong Luo1, Changchun

4 Zhou4, Zhou Xiang1, Pol Maria Rommens5, Ming Liu4*,and Ulrike Ritz5*

5 1Orthopedic Research Institute, Department of Orthopedics, West China Hospital,

6 Sichuan University, Chengdu, China.

7 2Department of Periodontology, Justus-Liebig-University of Giessen, Germany.

8 3LIMES Institute, Department of Molecular Brain Physiology and Behavior,

9 University of Bonn, Carl-Troll-Str. 31, 53115 Bonn, Germany.

10 4National Engineering Research Center for Biomaterials, Sichuan University,

11 Chengdu 610064, China

12 5Department of Orthopaedics and Traumatology, Biomatics Group, University

13 Medical Center of the Johannes Gutenberg University, Langenbeckstr. 1, 55131

14 Mainz, Germany

15 †Zhao Chen and Fei Xing contributed equally to this work and share the first

16 authorship.

17 * Corresponding author

18

19

20

21
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22 Abstract： DOI: 10.1039/D3TB00727H

23 Those who have used traditional biomaterials as bone substitutes have always

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24 regarded the immune response as an obstacle leading to implant failure. However,

25 cumulative evidence revealed that blindly minimizing host immune reactions cannot

26 induce successful bone regeneration. With the emergence of the new concept of

27 osteoimmunology, the intimate mutual effects between the skeletal system and the

28 immune system have been gradually recognized, promoting the innovation of

29 biomaterials with osteoimmunomodulatory properties. By tuning the surface properties,

30 biomaterials can precisely manipulate the osteoimmune environment favoring bone

31 regeneration. In this review, we first reviewed the mutual effects between the skeletal

32 system and the immune system to show the importance of immunomodulation on bone

33 regeneration. Subsequently, we summarize the recent developments in surface

34 modification strategies in terms of the surface physicochemical properties and surface

35 coatings and explain how these modification strategies work.

36 Keywords: bone repair, bone regeneration, osteoimmune, coating, bone healing.

37

38

39

40

41

42
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43 1. Introduction DOI: 10.1039/D3TB00727H

44 Bone regeneration is a precise process involving multiple systems. In the year of

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45 1972, Horton and co-workers first reported that stimulated lymphocytes were able to

46 induce bone resorption by secreating factor in organ cultures of fetal rat bones in vitro,

47 suggesting a potential correlation between the skeletal and the immune system [1]. In

48 1992, Donath et al. published their findings on the pathology of foreign body reactions

49 towards implanted materials in human organs [2], describing that interfacial bone is

50 formed as a result of foreign body reaction, once again confirming the relevance of the

51 immune response to bone regeneration. In the year of 2000, the concept of

52 osteoimmunology was proposed by Choi to describe the interdisciplinary phenomenon

53 in which T cells regulate osteoclastogenesis by maintaining the balance between

54 receptor activator of NF-kappaB ligand (RANKL) and interferon-gamma (IFN- γ )

55 produced by T cells [3, 4]. In the last two decades, this reciprocal regulation has been

56 evidenced by numerous shared molecules or signaling mechanisms identified between

57 both systems [5-8]. Previous studies have demonstrated that favorable bone

58 regeneration can be obtained by manipulating osteoimmunomodulation [9-11],

59 emphasizing the significance of the immunity for bone regeneration.

60 The management of bone defects is one of the most concerning and challenging

61 issues for clinicians [12, 13]. Recently, numerous biomaterials have been fabricated as

62 potential bone substitutes to promote bone regeneration, but they encounter host

63 responses due to their exogenous properties [14-16]. Typically, immediately after

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64 implantation, blood clots and proteins are absorbed on the biomaterial surfaces, further

65 dictating a cascade of events driven by the immune system that ultimately affects the

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66 outcome of bone regeneration [17-19]. By tuning surface properties, biomaterials can

67 precisely manipulate protein adsorption and the subsequent immune response, resulting

68 in favorable bone regeneration [14, 20-22]. Hence, numerous surface modification

69 strategies have been proposed, such as modification of surface morphology, roughness,

70 porosity, pore size, wettability, charge, functional groups, and coatings, aiming at

71 enabling biomaterials to shift the local immune environment from pro-inflammatory to

72 pro-healing.

73 In this work, we first reviewed the bone healing process to obtain a clear

74 understanding of the whole process. Then, we provide an overview of the mutual effects

75 between bone and immunity to show how these two systems interact and work together.

76 Subsequently, we outline the recent developments in applied surface

77 immunomodulatory strategies in terms of the surface physicochemical properties and

78 surface coatings, further explaining the possible underlying mechanisms. Such

79 strategies are capable of providing solutions for the challenges in bone tissue

80 engineering.

81 2. Normal bone union process

82 Bone union is a complex physiological process requiring participation of multiple

83 cells and molecules [23, 24]. Unlike other tissues, bone has an extraordinary ability to

84 heal without scarring [25, 26]. Generally, bone union mainly consists of four unique
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85 but overlapping phases: the inflammatory phase, soft callus formation, hard callus

86 formation and the bone remodeling phase [18, 27, 28] (Figure 1). Briefly, in the

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87 inflammatory phase, disruption of bone leads to the formation of hematoma around the

88 injured sites, serving as a temporal brace for immune cells infiltration. Bioactive factors

89 secreted by immune cells accelerate the recruitment of osteoprogenitor cells and

90 endothelial cells to the injury sites for further engagement in cartilaginous callus

91 formation. Subsequently, the soft callus begins to be absorbed and substituted by the

92 newly formed woven bone. Finally, in the remodeling phase, according to the Wolff

93 law, the woven bone is substituted by lamellar bone through continuous bone formation

94 and resorption, resulting in the restoration of the original bone structure [29].

95

96 Figure 1. The typical healing process of bone fracture.

97 3. Interconnections between bone and immune system

98 Bone marrow is the primary site of hematopoietic organs [30, 31]. Immune cells

99 derived from hematopoietic stem cells (HSCs) in the bone morrow share the same
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100 microenvironment as bone cells [6, 32]. Over the decades, with the advancements in

101 experimental methodologies, lots of researchers have attempted to utilize

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102 methodologies such as ELISA, gene expression analysis, and qualitative histology to

103 figure out the interactions between immune and bone system, and led to the generation

104 of ‘osteoimmunology’ [33-35]. For instance, by using ELISA, Christiansen et al. found

105 that the cytokine profile was significantly different in total hip replacements (THR)

106 patients experiencing aseptic loosening (AL) when compared with THR patients

107 without AL [36], suggesting the involvement of immunity in bone resorption. In

108 addition, in an in vivo study, Trindade and co-workers placed cp titanium into the distal

109 femurs of rabbits and demonstrated, for the first time by gene expression analysis, that

110 the use of titanium implants caused a significant increase in type 2 inflammation. At

111 the same time, histological studies confirmed that bone resorption was inhibited on the

112 titanium surface [37], suggesting an environment more conducive to bone formation.

113 Subsequently, Trindade et al. compared materials that do not osseointegrate (Copper

114 and Polyetheretherketone) to a material that osseointegrates (Titanium), describing the

115 immune reaction of each materials during bone healing [38]. The results showed that

116 both copper and polyetheretherketone exhibited a higher immune activity than titanium,

117 leading to a prolongation of the inflammatory phase during healing process, which

118 could be responsible for the failure of bone regeneration in contact with these materials.

119 Therefore, understanding these interactions will contribute to the successful preparation

120 of biomaterials with osteoimmunomodulatory properties. In this section, we will

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121 discuss the involvement of immune cells in bone regeneration and the modulation of

122 immune cells by bone separately.

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123 3.1 The involvement of immune cells in bone regeneration

124 Immune cells are demonstrated to participate in the whole process of bone healing

125 after injury [18]. Briefly, after the blood clot formation triggered by the initial damage,

126 neutrophils and polymorphonuclear leukocytes (PMNs) infiltrate the blood clot and

127 initiate the acute inflammatory phase of bone union. After that, attracted by cytokines

128 secreted by PMNs, the macrophages infiltrate the formed hematoma, exerting

129 osteogenic and angiogenic effect [39, 40]. Later, lymphocytes migrate into the callus

130 to initiate the adaptive immune response [41, 42].

131 Furthermore, studies have demonstrated that immune cells coordinate bone cells

132 to maintain bone homeostasis by balancing bone formation and bone resorption [43,

133 44]. For instance, previous studies found that the skeletal system and the immune

134 system share the same bone regulatory information via the

135 RANKL/RANK/osteoprotegerin (OPG) pathway [45, 46]. Briefly, RANKL is a ligand

136 needed for osteoclastgenesis, RANK is the receptor of RANKL, which is expressed by

137 osteoclasts and its precursors, and osteoprotegerin (OPG) is a decoy receptor for

138 RANKL [47]. Commonly, the ligation of RANKL by RANK leads to the activation of

139 nuclear factor kappa B (NF-kB) [48], nuclear factor of activated T cells 1 (NFATc1)

140 [49], and c-fos [49], causing osteoclast differentiation . OPG can interact with RANKL

141 to inhibit this pathway. In RA, T cells are demonstrated to express pro-inflammatory
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142 cytokines such as IL-17, IL-1, TNF-α, and IL-6 to induce the production of RANKL

143 from synovial fibroblasts, which contributes to the upregulated osteoclastic activity,

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144 resulting in bone destruction in RA [50, 51]. However, anti-inflammatory cytokines

145 such as IL-4 and IL-13 can inhibit bone resorption through the activation of receptors

146 on osteoblasts and osteoclasts, downregulating the levels of RANK in osteoclast

147 precursors and upregulating the levels of OPG in osteoblasts [52]. Moreover, recent

148 studies showed that B cells depletion may be effective in autoimmune diseases

149 including RA [53]. In-depth research found that B cells could secrete cytokines

150 including TNF-α, IL-6, and RANKL, participating in the bone destruction process in

151 RA [54, 55]. For instance, RANKL in B cells was thought to be able to facilitate

152 osteoclast differentiation, which could be triggered by B-cell receptor and CD40, and

153 further enhanced by IFN-γ secreted by T cells [56]. Taken together, the immune cells

154 are fully involved in balancing bone formation and bone resorption. An appropriate

155 activation of immune cells can balance bone dynamics that results in successful bone

156 formation. Biomaterials designed for bone regeneration should be focused on

157 modulation of immune microenvironment to direct bone cells towards bone formation.

158 3.2 Polarization of macrophage and its role in bone regeneration

159 The immune system is an interactive network of immune cells, organs, and

160 proteins known for its host defense ability [57-59]. Currently, its unique role in bone

161 repair has been gradually recognized. Among the immune cells, macrophages have

162 been studied most due to their capability of switching their phenotypes in response to
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163 varying inducers [60-62]. Generally, Macrophages can be categorized into M1 and M2

164 phenotypes. Macrophages of different phenotypes are relatively independent but

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165 closely connected. They participate through the whole process of bone regeneration as

166 a sweeper, a mediator, and an instructor [63, 64]. Briefly, as a sweeper, the

167 macrophages clear the foreign particles or debris through phagocytosis and remodel the

168 matrix after differentiation into osteoclasts [65]; As a mediator, the macrophages can

169 mediate the matrix mineralization and vacuolization through their paracrine cytokines

170 [66]. As an instructor, they can instruct the accumulation and maturation of osteoblast

171 [67]. In this situation, we will introduce the crosstalk among macrophages, osteoblasts,

172 and osteoclasts (Figure 2), and discuss its role in bone union process separately in

173 accordance with different phenotypes.

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174
175 Figure 2. The crosstalk among macrophages, osteoblasts, and osteoclasts.

176 For M1-polarized macrophages, which are known for the production of pro-

177 inflammatory cytokines, predominate in the early stage of acute inflammation [68, 69].

178 Briefly, under the attraction of inflammatory molecules, M1 cells infiltrate the injured

179 site, remove foreign particles or debris through phagocytosis, and in turn secrete pro-

180 inflammatory cytokines, namely, tumor necrosis factor alpha (TNF-α), interleukin 1

181 beta (IL-1ß) and interleukin 6 (IL-6), to initial inflammatory activity [70, 71]. Moreover,

182 bone is a highly vascularized tissue [72], blood vessels can supply oxygen, cells,

183 nutrients, and growth factors necessary for bone formation [46]. It has been reported
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184 that M1 cells participate in the formation of primitive vasculature by producing vascular

185 endothelial growth factor (VEGF), TNFα, and basic fibroblast growth factor (bFGF),

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186 suggesting the importance of M1 cells in bone formation (Figure 3). However, the long-

187 term presence of M1 cells may cause chronic inflammation and eventually nonunion of

188 bone defects [73, 74]. For instance, the study by Chan et al. found that a low dose

189 administration of TNF-α within 24 hours of injury augmented fracture repair in vivo

190 [75]. However, in another study, a persistent administration of TNF-α and IL-1β

191 resulted in impaired bone growth [76].

192
193 Figure 3. Macrophages in bone healing process. Macrophages are involved in all four healing

194 phases. M1 macrophages dominate the early stage of bone union process. During this stage, M1

195 cells participate in the initiation of acute inflammation and the formation of primitive vasculature

196 by producing cytokines including IL-1, IL-6, TNF-α and VEGF. In contrast, M2 macrophages

197 dominate the late stage of bone union process. They could promote bone regeneration and

198 participate in the late stage of angiogenesis by secreting cytokines including IL-10, TGF-β, BMP-2

199 and PDGF. The macrophages are also involved in the bone remodeling phase by balancing bone

200 formation and bone resorption.

201 For M2-polarized macrophages, which are known for the expression of anti-

202 inflammatory cytokines such as transforming growth factor beta (TGF-β), interleukin
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203 10 (IL-10), platelet derived growth factor (PDGF), and bone morphogenetic protein 2

204 (BMP-2), predominate in the late stage of bone union [68, 69, 77]. According to their

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205 functional differences, M2 cells can be further categorized into four subtypes [78]: M2a,

206 capable of stimulating fibroblasts to promote wound healing [79]; M2b, involved in

207 inflammatory modulation [80]; M2c, responsible for tissue remodeling [81]; and M2d,

208 extensively involved in angiogenesis [82] (Figure 4). By secreting anti-inflammatory

209 cytokines, M2 cells can inhibit bone resorption as a result of the enhanced expression

210 of OPG in osteoblasts. Furthermore, both TGF-β and BMP are members of the TGF

211 superfamily. As an upstream target of BMP signaling, TGF-β was demonstrated to

212 synergize with BMP to promote the osteogenic differentiation of MSCs through the

213 typical Smad-dependent signaling pathway and atypical Smad-independent signaling

214 pathway [60, 64, 65]. Simultaneously, M2 cells participate in the late stage of

215 angiogenesis by secreting platelet-derived growth factor (PDGF), serving as stabilizing

216 vasculature and anastomosing sprouting endothelial cells [83]. However, similar to M1

217 cells, long-term activation of M2 cells may increase the secretion of profibrotic

218 molecules and lead to excessive formation of scar tissues, thus delaying the healing

219 process [84] (Figure 4). Therefore, proper combination of macrophages with different

220 phenotypes and moderate activation are essential for successful bone regeneration, and

221 how to achieve this should be the direction of biomaterial scaffolds in the future.
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222
223 Figure 4. Schematic of macrophage polarization.

224 3.3 Regulation of the immune system by bone

225 Immune cells derived from HSCs in the bone morrow share the same

226 microenvironment as bone cells. Previous studies have found that bone cells

227 extensively participated in immune cells regulation [85, 86]. In this regard, we will

228 discuss the function of bone cells in immune cells regulation. And describe them

229 separately according to different bone cells (Figure 5).

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230

231 Figure 5. the regulatory role of bone cells in immune system.

232 3.3.1 Osteoblast regulation

233 Bone formation is closely coordinated with the normal functioning of bone

234 marrow hematopoiesis [87]. Cumulative evidence indicates that osteoblasts can

235 specifically secrete a variety of cytokines that are involved in hematopoietic stem cell

236 (HSC) regulation [88-91]. For instance, conditional ablation of osteoblasts in transgenic

237 mice resulted in a lower HSC number and markedly reduced B cells, which implied

238 that osteoblasts were able to support B-cell differentiation from HSCs. Further studies

239 have demonstrated that IL-7 and CXC motif chemokine 12 (CXCL12) secreted by

240 osteoblasts are implicated in this process [92]. Another study by Terashima et al. found

241 that ablation of osteoblasts or inducible deletion of IL-7 in osteoblasts led to

242 lymphopenia together with a loss of common lymphoid progenitors (CLPs).

243 Application of IL-7 saved the loss of CLPs. To assess whether immunodeficiency can

244 be corrected by augmenting osteoblasts, in vivo administration of parathyroid hormone

245 was employed to facilitate osteoblast formation, and an increased osteoblast number
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246 was observed to lead to an upregulation in IL-7 expression, resulting in ameliorated

247 lymphopenia [93]. Furthermore, osteoblasts also participate in immune cells regulation

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248 by secreting macrophage colony-stimulating factors (M-CSFs). Wiktor-Jedrzejczak et

249 al. confirmed that deficiency of M-CSFs in osteoporotic mutant mice led to a significant

250 reduction in macrophages and osteoclasts [94]. All these results demonstrated that

251 osteoblasts can be a potential target to manipulate immune cell behavior.

252 3.3.2 Osteoclast regulation

253 Osteoclasts are predominant effector cells that directly participate in the process

254 of bone resorption, providing enough cavity for immune cell maturation [6, 95].

255 Besides, the mutual effects of osteoclast on immune cells also exist in the autoimmune

256 diseases such as rheumatoid arthritis and multiple myeloma, manifested by increased

257 activity of osteoclasts and T cells [96, 97]. The study by Li et al. explained this

258 phenomenon by demonstrating that human osteoclasts can express Class I and Class II

259 major histocompatibility complex (MHC) molecules, as well as costimulatory

260 molecules, and induce both CD4+ and CD8+ T-cells responses [98], which suggests

261 that osteoclasts could function as antigen-presenting cells and induce regulatory T-cell

262 responses [98, 99]. In another study by Grassi et al., T cells was co-cultured with

263 osteoclasts to assess whether osteoclasts are immune competent cells. The osteoclasts

264 can hinder T-cell responses to PHA and CD3/CD28 stimulation, thereby restraining T-

265 cell proliferation, inhibiting T-cell TNF-α and IFN-γ expression, and decreasing T-cell

266 apoptosis [100]. In addition, Mansour et al. used zoledronic acid to establish
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267 osteopetrosis model in normal mice. As a results, downregulation of CXCL12 and IL-

268 7 expression in stromal cells resulted in a marked decrease in B cells, which suggests

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269 that osteoclasts can indirectly regulate the number of B cells by affecting osteoblasts

270 [101]. In summary, osteoclasts are extensively involved in the immune cells regulation,

271 making it possible to treat diseases involving both the skeletal system and the immune

272 system through the development of biomaterials.

273 3.3.3 Osteocyte regulation

274 Osteocytes, the most abundant cells in the skeleton, represent the terminally

275 differentiated state of the osteoblast lineage [102]. Cumulative evidence indicates that

276 osteocytes can regulate bone formation and remodeling through direct contact with

277 neighboring cells or by affecting cells in the bone marrow or distant organs through

278 paracrine and endocrine cytokines [103-105]. In this respect, several studies

279 documented the regulatory role of osteocytes on immune cells. For instance, Sato et al.

280 described that peripheral blood B and T lymphocytes were significantly decreased in

281 osteocyte-less mice, which was supposed to be related to the altered microenvironment

282 in primary lymphoid organs linked to the dysfunction of osteocytes [106]. Moreover,

283 Fujiwara et al. demonstrated that RANKL produced by osteocytes was necessary for

284 the increased osteoclasts and B cells in estrogen-deficient mice, suggesting that the

285 regulatory effect of estrogen on B cell numbers depended on osteocytes [107]. In

286 addition, sclerostin primarily secreted by osteocytes was documented to be vital for

287 supporting normal B-cell development using a sclerostin-knockout mouse model,

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288 which was speculated to be associated with the downregulation of CXCL12 expression

289 [108]. However, another study by Yee et al. concluded that osteoprogenitors and their

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290 progeny, rather than osteocytes, contributed most of the sclerostin to B cell

291 development [109], which suggests that despite some uncertainties in currently

292 available researches, it is undeniable that osteocytes have a regulatory role in the

293 immune system.

294 4. Surface physical properties design based on osteoimmune modulations for bone

295 regeneration

296 Recently, the mutual effects of biomaterials on immune cells have been gradually

297 recognized, providing new directions to fabricate bioactive scaffolds. Generally, the

298 optimal biomaterials are thought to be a collection of properties, including

299 immunomodulation, pro-angiogenesis, and pro-osteogenesis. In the last few decades, it

300 has been well established that alterations in the surface properties can alter the immune

301 microenvironment to favor osteogenesis and angiogenesis.[44, 110-114]. Changes of

302 the surface physical properties influence the adsorbed layer of proteins and subsequent

303 signal transduction, resulting in altered cellular behavior of the immune cells [114].

304 Briefly, changes in biomaterial surface properties can modulate the adhesion, activation

305 and fusion of immune cells [115-117]. In consideration of these, strategies to modulate

306 surface physical properties have received more attention. A growing number of scholars

307 have attempted to manipulate the immune microenvironment by modifying the physical

308 properties of the biomaterial surface, including topography, roughness, porosity and
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309 pore size (Table 1), to achieve successful osseointegration. That is, direct contact

310 between living bone and implant at the light microscope level [118].

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311 Table 1. Effects of surface physical properties on immunomodulation.

Physical properties Feature Immunomodulatory effects Ref.

Topographies Tubes: Attenuate the activity of macrophage-related [119]

78nm in diameter inflammatory reactions
Dots: The expression levels of pro-inflammatory genes in [120]
10~200 nm in diameter macrophages were remarkably upregulated on the
100~200 nm surface
Fibers: Downregulated the pro-inflammatory cytokines in [121]
0.55±0.16 µm in diameter macrophages
Grooves: Downregulated the pro-inflammatory cytokines by [122]
25–30 µm in groove structure macrophages
Pores: Pore sizes influence the shape of macrophages and the [123]
15~200nm in pore sizes subsequent autophagy pathway, the osteogenesis was
promoted most on the 50nm surface.
Rods: Nanorods defined as HA-100 accelerated osteogenesis [124]
HA-0: 20–30 nm(length), 5–10 nm(width) most through promoting the expression of T Cell-Derived
HA-30: 30–40 nm(length), 13–15 nm(width) IL-22
HA-100: 45–70 nm(length), 10–15 nm(width)
Roughness Microscale roughness: Upregulated the expression of anti-inflammatory [125]
Sa: 3.64±0.029 μm for modSLA cytokines by macrophages.
Sa: 3.61±0.047 μm for plasmaSLA
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Sa: 0.59±0.023 μm for plasmaPT
Nanoscale roughness: Downregulation of the inflammatory response was [126]
Sa: 1.50±0.15 µm, observed
Sdr: 45.52±8.98%
Grooves: Polarization of macrophages toward the prohealing [127]
200 nm-50 µm phenotypes was the greatest on 400-500 nm surface
Porosity and pore size Porosity Higher porosity scaffolds exhibited immunomodulatory [128]
34.4±1.3% vs 14.5±0.8% properties
Pore size: P600 promoted M2 polarization in macrophages most [129]
P200: 209.9±77.1µm and significantly reduced the foreign body response
P400: 385.5±28.6 µm
P600: 582.1±27.2 µm
312 Sa: average roughness over area; Sdr: developed interfacial area ratio.
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313 4.1 Topography design

314 Modification of biomaterial surface topographies have been documented to be

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315 valid in adjusting the immune cell response, facilitating the formation of an immune

316 microenvironment for better osseointegration [130-132]. Generally, the surface

317 topography is determined by surface orientation and roughness. In brief, surface can be

318 classified into anisotropic surface and isotropic surface, depending on whether they

319 have a clear orientation or not. Simultaneously, in accordance with surface roughness,

320 surface can be classified into micrometre surface and nanometre surface. Different

321 manufacturing processes will generate various orientations and roughness [133]. In the

322 past, the primary machining techniques used to alter surface topography include

323 electropolishing, mechanical polishing, blasting, etching, oxidation, coatings, plasma

324 spraying, and ion deposition. Today, a large variety of machining techniques have been

325 introduced for the processing of material surface topography, such as photolithography,

326 electron beam and X-ray lithography, phase separation, colloidal self-assembly,

327 electrospinning, and pattern transfer. Advances in manufacturing technology have

328 facilitated the development of biomaterials with controllable topographical features

329 [134], which are thought to play an important role in the process of

330 osteoimmunomodulation [135]. Currently, numerous morphologies of surface

331 topographies, such as tubes, colloids, grooves, pillars, fibers and dots, have been

332 produced in biological research (Figure 6). These surface topographies can be both at

333 the micron and nanoscale. Most of the previous studies concluded that the effect of
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334 surface topography on osteogenesis occurred mainly at the micron level. However, as

335 research has progressed, researchers have realized that there is no close correlation

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336 between micrometre structures and nanometre structures, and the role of nanostructures

337 in the process of bone formation cannot be ignored [136]. To correctly understand the

338 relationship between topography and bone formation, the study of material surface

339 nanotopography is crucial. Despite the fact that nanometre structures have likewise

340 been detected on some commercial implants, but studies on nanotopography are still

341 focused on in vitro experimental studies, clinical studies on nanotopography are still

342 lacking. In this section, we will discuss the effect of different morphology of surface

343 topography on immune response and osteogenesis. And the discussion of surface

344 topography roughness will be continued in the next section. The findings, including

345 immunomodulatory properties or the capabilities to promote osteogenesis, are

346 summarized below.

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347
348 Figure 6. Schematic representation of typical nanostructures for bone regeneration applications.

349 Reprinted with permission [44]. Copyright 2017, Royal Society of Chemistry.

350 In an in vitro study conducted by Neacsu P et al., macrophages were co-cultured

351 with different implant surfaces to investigated the mutual effects between topography

352 and immune system. The results showed that TiO2 tubes with a diameter of 78 nm could

353 attenuate the activity of macrophage-related inflammatory reactions to obtain a

354 response favoring healing process when compared with CP titanium surfaces [119]. In

355 a subsequent study, Neacsu and colleagues used ELISA to study the possible

356 mechanism of the immunomodulatory role of nanotubes in vitro. The results showed

357 that the nanotubes may attenuate the macrophage-related inflammation by inhibiting

358 the mitogen-activated protein kinase (MAPK) and the nuclear factor kappa-light-chain-
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359 enhancer of activated B cells (NF-kB) pathways [137]. In a study conducted by

360 Mohiuddin et al., the effect of surface topography on macrophage behavior was studied

Journal of Materials Chemistry B Accepted Manuscript

361 by culturing macrophage on nanodot surfaces with different parameters in an in vitro

362 assay [120]. As the results showed, an increase in macrophage adhesion was noted on

363 nanodot arrays with diameter ranging from 10 to 50 nm, while decreased cell adhesion

364 was observed on the 100-200 nm surface. Simultaneously, a significantly upregulated

365 expression of pro-inflammatory genes was observed in macrophages on the 100-200

366 nm surface, suggesting the possible immunomodulatory roles of nanodots ranging from

367 10-50 nm. In an in vitro study performed by Saino et al., fibers possessing different

368 diameter were fabricated on scaffold surfaces by using electrospinning technique.

369 Macrophages were then seeded on different surfaces to investigate the influence of fiber

370 diameter on macrophage polarization. The ELISA and histological results showed that

371 nanofibrous scaffolds (Diameter: 0.55 ± 0.16 um) minimized the inflammatory

372 response when compared with films and microfibrous scaffolds (1.60 ± 0.25 μm) [121]

373 (Figure 7). In an in vitro study performed by Li et al., in order to imitate the structure

374 of natural bone matrix, 25–30 μm groove structure was fabricated on the surface of

375 hydroxyapatite scaffolds (HAS) to construct micro-grooved HAS (G-HA).

376 Macrophages were then cultured on G-HA to assess the influence of groove structure

377 on immune environment, ELISA results showed that the expression of pro-

378 inflammatory cytokines in macrophages was significantly decreased in the G-HA group

379 compared to the HAS group. Moreover, enhanced osteogenic differentiation of BMSCs
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380 was observed in macrophage/G-HA conditioned medium, which was demonstrated in

381 subsequent experiments to be a possible consequence of decreased IL-6 expression in

Journal of Materials Chemistry B Accepted Manuscript

382 macrophages causing downregulation of miR-214 and subsequent upregulation of the

383 p38/JNK pathway [122]. In another study conducted by Yu et al., bone-mimicking HA

384 nanorods with different aspect ratios were placed into mandibular defect in mouse to

385 investigate the effect of the nanometre structures on osteogenesis. Unlike other studies,

386 this is an in vivo study. The outcomes of imaging and histological tests showed that

387 nanorods with lengths of 45–70 nm and widths of 10–15 nm could accelerate

388 osteogenesis most. In further in vitro experiments, the accelerated osteogenesis was

389 demonstrated to be related to the upregulated expression of T Cell-Derived IL-22 [124],

390 suggesting the involvement of innate immune response during bone regeneration

391 process. It remains unclear how surface topography is involved in

392 osteoimmunomodulation and limited evidence suggests that this may be related to

393 altered macrophage morphology. For instance, Chen et al. performed an in vitro study

394 aiming to evaluate the role of immune cells on topography-mediated osteogenesis.

395 Macrophages were seeded on the nanoporous surfaces with different pore sizes. As a

396 result, different cell morphologies and affinities were observed on different surfaces,

397 leading to the differences in the activation of macrophage autophagy and subsequent

398 anti-inflammatory reaction. Thereafter, the differences in anti-inflammatory responses

399 were demonstrated to lead to differences in the activation of osteogenic pathways. This

400 result suggested that the regulation of macrophage shape and subsequent autophagy
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401 may be crucial for the conversion of graphical information to biological information

402 [123]. Although numerous experimental studies have identified the ability of surface

Journal of Materials Chemistry B Accepted Manuscript

403 topography to modulate immunity and alter the osteogenic process. It is worth noting

404 that most of the present outcomes are derived from in vitro studies and should be

405 considered as hypothesis-generating. Animal experiments and clinical studies are

406 needed to elucidate the underlying mechanisms on the osteoimmunomodulatory roles

407 of surface topographies. At the same time, we need to be aware of that excessive bone

408 regeneration at the interface may not be a clinical advantage, re-emphasizing the critical

409 importance of in vivo studies for the development of successful bone regeneration

410 materials.
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Journal of Materials Chemistry B Accepted Manuscript

411
412 Figure 7. The role of topography in osteoimmunomodulation. (A) Schematic of the influence of

413 electrospun fiber diameter on the inflammatory response. Nanofibrous PLLA scaffolds alleviated

414 the inflammatory response compared to the others. (B) Cytokines secreted by RAW 264.7 cell after

415 7 days of culture on scaffolds. Relatively lower pro-inflammatory cytokines were released by

416 macrophage cells cultured on nanofibers compared to the films and microfibrous scaffolds. (A-B)

417 Reprinted with permission [121]. Copyright 2011, American Chemical Society. (C) SEM and

418 fluorescent images of BMDMs cultured on Ti substrate with various groove sizes. The elongation

419 of macrophage was most prominent on grooved surfaces of 450 nm in width. (D)

420 Immunofluorescent images and quantification of Arg1 (anti-inflammatory marker) expression of

421 macrophages on control and grooved substrates. Arg1 expression was upregulated on grooved

422 surfaces ranging from 400 nm to 5 μm. (C-D) Reprinted with permission [127]. Copyright 2015,

423 American Chemical Society.

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424 View Article Online

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425 4.2 Roughness design

Journal of Materials Chemistry B Accepted Manuscript

426 Surface roughness has been well established as a modification method to improve

427 osseointegration, and it has been reported to be capable of regulating the osteoimmune

428 environment to accelerate bone regeneration [138]. Generally, accordance to the

429 surface roughness, surface can be classified into micrometre surface and nanometre

430 surface Actually, it has been demonstrated that an oral implant called TiUnite

431 possessing moderately rough surface at the micron level of resolution presents more

432 desirable results under challenging clinical conditions when compared to other

433 moderately rough surfaces, minimally surfaces, and rough surfaces [139]. Similar

434 results were also found in an in vitro study conducted by Hotchkiss et al., who evaluated

435 the effect of microroughness on cell behavior by culturing macrophages on rough

436 surfaces (Sa: 3.64±0.029 μm for modSLA; Sa: 3.61±0.047 μm for plasmaSLA) or

437 smooth surface (Sa: 0.59±0.023 μm for plasmaPT). The results showed that the

438 expression of anti-inflammatory cytokines on rough surfaces was significantly higher

439 than that on smooth surfaces, suggesting that rough surfaces could control inflammation

440 and improve the success of implanted materials [125]. Today, nanoscale surfaces have

441 been researched in frequent to imitate the roughness of natural bone. However, clinical

442 evidence supporting the use of any nanoscale surfaces is still lacking, at best, is based

443 on the results of animal studies [140]. Limited researches suggested that nanoscale

444 surface modifications in implants could favorably influence molecular and cellular

445 behavior, leading to altered bone regeneration [141]. In an animal study performed by
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446 Thalji et al., titanium implants possessing nanosurface features (Sa: 1.50±0.15 µm, Sdr:

447 45.52±8.98%) and microroughened surfaces (Sa: 1.58±0.16 µm, Sdr: 66.01±10.43%)

Journal of Materials Chemistry B Accepted Manuscript

448 were placed in the tibiae of rats, respectively. Subsequently, whole genome microarray

449 was utilized to obtain the gene expression profiles of cells adherent to these two

450 implants. In this study, the nanosurface features did not have a significant effect on

451 surface roughness on a micrometer level. An earlier amelioration of the inflammatory

452 response was detected on the nanoscale surfaces compared to the microroughened

453 surfaces. Furthermore, a faster and higher osseointegration was shown on the

454 nanosurface than on the microroughened surface, as revealed by the upregulation of

455 genes expressed in the process of osteogenic differentiation [126]. In an in vitro study,

456 Luu et al. designed surfaces with grooves of varying widths (200 nm-50 µm) on

457 titanium substrates, aiming to examine the regulatory role of surface roughness in

458 immune cells. The results revealed that macrophage elongation was observed to be the

459 greatest on surfaces with grooves ranging from 400 to 500 nm in width, resulting in the

460 polarization of macrophages toward the prohealing phenotypes [127] (Figure 7). In

461 summary, roughness is an important parameter of scaffold surfaces, and surfaces with

462 different roughness can induce macrophages to be polarized towards different

463 phenotypes. There is still a need to investigate how the altered implant surfaces

464 influence immune cell behavior, leading to alterations in the bone regeneration process.

465 Moreover, more clinical evidence is needed to determine the appropriate surface

466 roughness in the process of osseointegration.

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467 4.3 Porosity and pore size design

468 Porosity and pore size are considered to be important surface properties since

Journal of Materials Chemistry B Accepted Manuscript

469 porous structure is closely related to bone regeneration [142, 143]. The porous structure

470 is essential for nutrition exchange and waste removal, and the interconnected networks

471 provide guidance for cell migration and tissue formation [144]. Porosity is one of the

472 major parameters that are supposed to be taken into consideration during the

473 manufacturing process of a scaffold. Higher porosity appears to be associated with

474 enhanced cell proliferation and successful osseointegration; however, excessively high

475 porosity can reduce the mechanical strength of biomaterials, leading to implant failure

476 [144]. For instance, a study performed by Rnjak-Kovacina et al fabricated synthetic

477 human elastin scaffolds with varying porosities (34.4±1.3% vs 14.5±0.8%) by changing

478 the flow rates during electrospinning, and only scaffolds with higher porosity facilitated

479 cell migration and infiltration in vitro. Moreover, scaffolds with higher porosity

480 sustained for at least 6 weeks in vivo and did not elicit any excessive immune response,

481 suggesting that higher porosity may confer favorable immunomodulatory properties to

482 the substrates [128]. In addition, the pore sizes are equally vital for an optimal scaffold

483 system. Commonly, smaller pores could cause a local hypoxic environment leading to

484 enhanced inflammatory reactions, resulting in the formation of inflammatory

485 granulation that blocks the mutual effects between the surrounding environment and

486 the implants [145]. To date, however, there is no recommended standard for pore size.

487 Generally, scaffolds with apertures greater than 300 µm are recommended to promote
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488 bone regeneration [142]. For instance, Li et al. designed a suite of bioactive scaffolds

489 with various pore sizes by using a pneumatic extrusion 3D printer, then scaffolds with

Journal of Materials Chemistry B Accepted Manuscript

490 various pore sizes were implanted into the subcutaneous models and mandibular critical

491 bone defect models in SD rats, respectively. In vivo testing found that the scaffolds

492 with an average pore size of 582 μm significantly reduced the foreign body response in

493 comparison to others with smaller pores. In addition, more M2 polarization,

494 angiogenesis, and bone regeneration were also detected on this set of scaffolds, and

495 further research confirmed that MyD88 protein was supposed to be implicated in the

496 regulation of macrophage polarization at various pore sizes [129] (Figure 8). Similar

497 findings were also reported by Garg et al., who found that higher porosity or larger

498 pore size of scaffolds were able to promote macrophages polarization toward the M2

499 phenotype in vitro [146]. These results suggest that the porosity and pore size are key

500 physical properties of biomaterials, endowing implants with the ability to modulate the

501 immune environment and integrate bone tissue. Determining the optimal physical

502 parameters that balance biological performance, mechanical strength and

503 biodegradability remains the focus of future research.

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Journal of Materials Chemistry B Accepted Manuscript

504
505 Figure 8. Immunomodulatory effect of pore size in biomaterials. (A) SEM images of

506 polycaprolactone/polyethylene glycol/hydroxyapatite scaffolds of various pore sizes, including

507 P200 (209.9 ± 77.1 μm), P400 (385.5 ± 28.6 μm), P600 (582.1 ± 27.2 μm). (B) images of MyD88

508 immunohistochemical staining for scaffolds after implantation in rat bone defects. The P600 group

509 showed lower MyD88 protein expression, which has been demonstrated to participate in regulating

510 macrophages polarization toward M2 phenotype. (C) Fluorescent images of macrophage

511 polarization markers for scaffolds after subcutaneous implantation in rats. The P600 group

512 promoted macrophages polarization toward the anti-inflammatory phenotype. (D) Fluorescent

513 images of macrophage polarization markers for scaffolds after implantation in rat bone defects. The

514 P600 group promoted macrophages polarization toward M2 phenotype. (A-D) Reprinted with

515 permission [129]. Copyright 2022, American Chemical Society.

516 5. Surface chemical properties design based on osteoimmune

517 modulations for bone regeneration

518 Surface chemistry alterations are strongly related to the adsorption layer of the

519 proteins [147]. Previous studies have confirmed that the surface interaction between the

520 substrate and the adsorption layer of the proteins is critical in determining the immune

521 response toward the implant [148, 149]. Therefore, the immune environment can be
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522 modulated by changing the surface chemistry of the scaffolds, such as the wettability,

523 surface charge or functional groups (Table 2).

Journal of Materials Chemistry B Accepted Manuscript

 Journal of Materials Chemistry B Page 34 of 71

Journal of Materials Chemistry B Accepted Manuscript

524 Table 2. Effects of surface chemical properties on immunomodulation.
Chemical properties Feature Immunomodulatory effects Ref.
Wettability Hydrophilic surfaces: plasmPT, plasmSLA and Hydrophilic surfaces promoted M2 polarization in macrophages and [125]
modSLA (contact angles: 0°) upregulated the production of IL-4 and IL-10
Hydrophobic surfaces: PT, SLA and aged modSLA
(contact angles: 93.6°, 120.9°, 110.4°, respectively)
GO-modified SLA vs SLA: (contact angles: 34.6±2.4° Enhanced M2 polarization in macrophages was observed on the GO- [150]
vs 97.0±1.6°) modified SLA surface as compared to the raw surface
Surface charge Surface with different charge: In contrast to the cationic surface, the anionic surface induced an [151]
Anionic surfaces anti-inflammatory response
Cationic surfaces
Surface with different potentials: T–P2 sample was more capable of inducing M2 polarization than the [152]
Polydopamine modified titanium surface (T-P1) vs T–P1 and T samples
thermally treated T-P1 (T-P2) vs titanium surface (T)
(zeta potentials: -80 vs -100 vs -40 mV)
Functional group Surfaces treated by oxygen or nitrogen plasma The production of pro-inflammatory genes in macrophages were [117]
upregulated in the presence of oxygen plasma treatment
Nanoparticles carrying different functional groups Macrophages cultured on nanoparticles modified with amide, [153]
(amide, epoxide, alkene, ether, ketal, nitro, or oxime) epoxide, and alkene tended to polarize toward the M2 phenotype.
Surfaces modified by NH2 and COOH groups Surfaces functionalized with NH2 and COOH groups induced pro- [154]
and anti-inflammatory macrophage responses, respectively.
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526 5.1 Wettability design

527 Hydrophobic surfaces exhibit intrinsic immunogenicity compared to hydrophilic

Journal of Materials Chemistry B Accepted Manuscript

528 surfaces, which may be related to differences in adsorbed proteins [155]. Usually,

529 proteins bind more readily to hydrophobic surfaces than to hydrophilic surfaces

530 because of hydrophobic interactions [156-158]. Furthermore, a previous study has

531 reported that the adsorbed proteins can induce inflammation and foreign body reactions

532 through conformational changes [148]. Therefore, it may be possible to modulate the

533 adsorbed layer of proteins and subsequent immune responses by altering surface

534 wettability. For instance, an in vitro study aiming to investigate the effect of material

535 surface wettability on macrophages polarization and cytokines expression was

536 performed by Hotchkiss et al., macrophages were cultured on titanium surfaces with

537 varied wettability, and hydrophilic surfaces treated by oxygen plasma cleaning were

538 demonstrated to induce polarization of macrophages towards M2 phenotype and

539 upregulate IL-4 and IL-10 expression [125]. Similarly, an in vitro study by Lv et al.

540 reported that macrophages on hydrophilic surfaces tended to polarize toward the M2

541 phenotype compared with macrophages on hydrophobic surfaces. Further mechanistic

542 studies have demonstrated that surface wettability may affect the adsorption and

543 subsequent conformational changes of specific proteins, leading to the selective

544 expression of integrin β 1 or β 2, which in turn activates different signaling pathways

545 (PI3K and NF-κB) and ultimately affects the behavior of macrophages [159] (Figure

546 9). In another in vitro study by Li et al., graphene oxide (GO) was deposited on the
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547 sandblasting and acid etching (SLA) surface using the ultrasonic atomization spraying

548 technique. Then macrophages and BMSCs were culture on the surfaces, respectively.

Journal of Materials Chemistry B Accepted Manuscript

549 The GO-modified surfaces exhibited higher hydrophilicity compared to the SLA

550 surfaces. Both M2 polarization of macrophages and osteogenic differentiation of

551 BMSCs were improved on the GO-modified surfaces, suggesting a potential role of

552 surface hydrophilicity in osteoimmunomodulation [150] (Figure 9). It is worth noting

553 that excessive augmentation in surface hydrophobicity may lead to high protein

554 resistance, reducing the immunomodulatory effects of biomaterials, which in turn is

555 detrimental to the clinical application of the materials [145]. Furthermore, In vivo

556 evidence is still lacking, future strategies lie in identifying appropriate surface

557 wettability through in vivo studies to regulate the immune response in the direction of

558 promoting osseointegration.

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Journal of Materials Chemistry B Accepted Manuscript

559
560 Figure 9. Effect of surface wettability on osteoimmunomodulation. (A) Schematic of the possible

561 mechanisms of surface hydrophilicity on macrophage behavior. Reprinted with permission [159].

562 Copyright 2018, John Wiley and Sons. (B) Schematic description of dual roles of GO-modified

563 titanium surface. The GO-modified surfaces exhibited higher hydrophilicity compared to the SLA

564 surfaces. Enhanced M2 polarization of macrophages on surfaces after GO modification provided a

565 favorable immune microenvironment for the success of osseointegration. Adapted from ref. 143

566 [150].

567 5.2 Surface charge design

568 The surface charge of biomaterials has been widely investigated for its capability

569 to mimic the bioelectric microenvironment as well as its role in immunomodulatory

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570 processes [114, 160]. Previous studies have confirmed that a charged surface can

571 influence the concentration and conformation of adsorbed proteins and subsequent cell

Journal of Materials Chemistry B Accepted Manuscript

572 adhesion [110, 161]. For instance, the study by Ohgaki et al. has described this process.

573 Briefly, calcium ions first bind to the negatively charged surface by electrical attraction,

574 then calcium ions attract cell adhesion proteins to adsorb on the scaffold surface, and

575 eventually affect osteoblasts adhesion and proliferation [162]. Moreover, the charged

576 surfaces were also thought to affect the immune environment surrounding biomaterials.

577 previous study reported that cationic particles can promote inflammation more than

578 anionic particles [151]. Numerous studies have attempted to modulate macrophage

579 polarization by altering the surface charge of biomaterials. Brodbeck et al. investigated

580 the effect of surface charge on cytokine production using an in vitro monocyte or

581 macrophage culture system, and the results showed that IL-10 expression in monocytes

582 or macrophages was significantly upregulated on anionic surfaces but downregulated

583 on cationic surfaces. Conversely, a significant reduction of IL-8 was noted in cells on

584 anionic surfaces [151], suggesting that anionic surfaces can induce anti-inflammatory

585 responses by regulating the expression of cytokines in monocytes or macrophages. In

586 addition, the potential intensity was reported to be equally vital in the process of

587 immunomodulation. For instance, Li et al. used a polydopamine coating to modify the

588 titanium surface to reduce its surface potential. In vitro, bone marrow-derived

589 monocytes (BMDMs) were seeded on samples with different surface charge and the

590 supernatant was collected to treat a mouse embryo cell line (C3H10T1/2). In vivo,
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591 samples were implanted into the mice air-pouch models and rats femur defect models,

592 respectively. Both in vitro and in vivo studies reported a tendency for macrophages to

Journal of Materials Chemistry B Accepted Manuscript

593 differentiate towards the M2 phenotype on low-potential titanium surface. Whole gene

594 expression analysis revealed that this may be related to the inhibition of PI3K-Akt-

595 mTOR signaling axis. Furthermore, both in vitro and in vivo studies indicated that

596 osteogenic differentiation of C3H10T1/2 was enhanced by the cytokines secreted by

597 M2 macrophages [152] (Figure 10). These findings suggest that the surface charge

598 regulation, including surface potential, may prove to be a viable solution for the

599 successful development of bone tissue engineering materials with immunomodulatory

600 properties.

601
602 Figure 10. Effect of surface charge on osteoimmunomodulation. (A) Effect of surface potential on

603 macrophage polarization and subsequent osseointegration. Implants with low surface potential promoted

604 the M2 cells polarization and subsequent bone regeneration. (B) Fluorescent images of macrophage

605 polarization markers in tissues surrounding different implants. Implants with lower surface potential
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606 were more capable of alleviating inflammatory reactions in vivo. (C) Schematic figure of DOI:
signaling
10.1039/D3TB00727H

607 pathways that influence macrophages polarization. Higher electron repulsion between low-potential

Journal of Materials Chemistry B Accepted Manuscript

608 titanium surface and bone marrow-derived monocytes (BMDMs) can promote the expression of integrin

609 β1 and integrin β3 in the cell membrane through the focal adhesion kinase signaling pathway, thereby

610 promoting the polarization of BMDMs toward M2 phenotype. The inhibition of PI3K-Akt-mTOR signal

611 axis was involved in this process (A-C) Reprinted with permission [152]. Copyright, 2022 Elsevier.

612 5.3 Functional group design

613 Surface functional groups can also affect the adsorption of proteins and subsequent

614 cellular responses [163, 164]. In this respect, several studies reported that surface

615 functional groups could influence immune cell behavior and subsequent new bone

616 formation. For instance, in an in vitro study conducted by Ion et al., oxygen or nitrogen

617 plasma treatment was employed to enrich the functional groups on the surfaces of

618 carbon nanowalls and investigate their influence on macrophage behavior. As the SEM

619 showed, oxygen plasma significantly promoted macrophage spreading and adhesion.

620 In addition, the expression levels of pro-inflammatory cytokines such as tumor necrosis

621 factor-alpha (TNF-α) and macrophage inflammatory protein-1 alpha (MIP-1α), were

622 significantly higher after oxygen plasma treatment compared with nitrogen plasma

623 treatment, suggesting that changes in the functional groups may affect the polarization

624 direction of macrophages [117]. In an in vivo study conducted by Bygd et al.,

625 nanoparticles carrying different functional groups were injected into SKH1-E mice to

626 investigate the influence of surface chemistry on macrophage polarization, in vivo

627 results revealed that macrophages that were adjacent to nanoparticles modified with

628 amide, epoxide, and alkene tended to polarize toward the M2 phenotype. Instead,
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629 macrophages adjacent to nanoparticles modified with ether, ketal, nitro, or oxime were

630 inclined to polarize toward the M1 phenotype [153], confirming the potential ability of

Journal of Materials Chemistry B Accepted Manuscript

631 surface modifications to alter macrophages phenotype in vivo (Figure 11). In another

632 study conducted by Buck et al., NH2 and COOH groups were attached on the surface

633 of poly(etheretherketone) (PEEK) implants via a diazonium-based chemical reaction to

634 solve the problems of poor regeneration of PEEK implants because of chronic

635 inflammation. In vitro study found that surfaces functionalized with NH2 and COOH

636 groups induced pro- and anti-inflammatory macrophage responses, respectively.

637 Meanwhile, COOH surfaces, which was able to induce an anti-inflammatory response,

638 significantly promoted the osteogenic differentiation of MSC compared with NH2

639 surfaces. However, the most deposition of calcium phosphates were observed on NH2

640 surfaces rather than on COOH surfaces after incubation in simulated body fluid for 3

641 weeks. For further validation of the osseointegration ability of the implants, samples

642 were implanted into the tibia unicortical defect models in rats and subsequently

643 subjected to CT and histological examination. In vivo study showed that both NH2 and

644 COOH surfaces were in contact with more new bone when compared with unmodified

645 surfaces. especially for the NH2 surfaces. At the same time, most increased bone-

646 bonding was detected for NH2 surfaces. Therefore, mineralization, osteogenesis and

647 immunomodulation are all important for osseointegration, successful implants need to

648 take these factors into account and be validated by careful in vivo testing [154] (Figure

649 11). In summary, modification of the surface functional groups may be a potential way
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650 to regulate the immune response and subsequent osseointegration process. However,

651 more in vivo studies are needed to balance the relationship between mineralization,

Journal of Materials Chemistry B Accepted Manuscript

652 osteogenesis and immunomodulation.

653
654 Figure 11. Effect of surface functional group on osteoimmunomodulation. (A) Schematic of influence

655 of surface functional groups on mineralization, macrophage polarization and osseointegration. Surfaces

656 functionalized with NH2 and COOH groups induced pro- and anti-inflammatory macrophage responses,

657 respectively. Meanwhile, Meanwhile, COOH surfaces, which was able to induce an anti-inflammatory

658 response, significantly promoted the osteogenic differentiation of MSC compared with NH2 surfaces.

659 However, more mineral-binding were observed on NH2 surfaces rather than on COOH surfaces,

660 suggesting that PEEK implant integration may be improved with mixtures of these two functional groups.

661 Reprinted with permission [154]. Copyright 2022, American Chemical Society. (B) Fluorescent images

662 of macrophage polarization markers in tissues surrounding different implants. Macrophages adjacent to

663 nanoparticles modified with epoxide tended to polarize toward the M2 phenotype. Instead, macrophages

664 adjacent to nanoparticles modified with nitro were inclined to polarize toward the M1 phenotype.

665 Reprinted with permission [153]. Copyright 2015, Elsevier.

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666 6. Surface coatings design based on osteoimmune modulations for DOI: 10.1039/D3TB00727H

667 bone regeneration

Journal of Materials Chemistry B Accepted Manuscript

668 To our knowledge, immune response is inevitable to any biomaterial. The ability

669 of immune system to shield off implants to protect nearby tissues is an important part

670 of its response to biomaterials [140]. Implants are constantly subjected to a number of

671 factors that may threaten their effectiveness, such as poor techniques, patient genetic

672 characteristics, local microorganisms, certain pharmaceutical products, corrosion etc.,

673 which may cause immune system-based rejection of implants. Such rejections are

674 limited to less than 10% at 25 years follow up after oral and orthopedic implants [165].

675 Apparently, oral and orthopedic implants function relatively well without any coatings.

676 But any implant is in constant threat of being rejected by the immune system

677 nevertheless. Whether actually coating materials are beneficial to improve clinical

678 function is unknown, but it is certain that this possibility exists [166, 167]. In the last

679 decades, several studies on implant coatings have demonstrated that the immune

680 microenvironment favoring osteogenesis seems to be closely associated with enhanced

681 osseointegration on coated surfaces [168-170]. In this section, we will summarize a

682 range of surface coatings that exhibit favorable immunomodulatory properties and

683 make some recommendations for the future development of implant coatings (Table 3).
 Journal of Materials Chemistry B Page 44 of 71

Journal of Materials Chemistry B Accepted Manuscript

684 Table 3. Effects of surface coatings on immunomodulation

Surface coatings Feature Immunomodulatory effects Ref.

Hydroxyapatite coatings HA coatings Upregulated the levels of anti-inflammatory cytokines [171]

HA nanoparticles coatings Enhanced the M2 macrophage polarization [172, 173]
Bioactive glass coatings Nanometer-sized bioactive glass Altered the inflammatory cytokines profile (low TNFα, high IL-1β), [174]
which has been further demonstrated to be beneficial for bone formation
Bioactive glass S53P4 coatings Presented excellent host-implant immune compatibility [175]
Extracellular matrix coatings Enamel matrix derivate Reduced the secretion of pro-inflammatory cytokines [176]
Glycopeptide hydrogel mimicking Enhanced the M2 macrophage polarization [170]
natural extracellular matrix
Metal ions coatings Magnesium ions Stimulated the macrophages polarization toward the anti-inflammatory [168, 177,
phenotype 178]
Strontium ions Ameliorated inflammatory infiltration [179, 180]
Anti-inflammatory cytokines IL-4 Stimulated the selective M2 polarization of macrophages [181-183]
loaded coatings
Drug-loaded coatings Indomethacin and tannic acid Downregulated the pro-inflammatory cytokines and upregulated the [167]
anti-inflammatory cytokines,
Aspirin Attenuated inflammatory response [184]
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686 6.1 Hydroxyapatite coatings

687 HA has been widely regarded as an ideal bone substitute for its satisfactory

Journal of Materials Chemistry B Accepted Manuscript

688 osteoconductivity, stable chemical properties and capability to facilitate angiogenesis

689 [185-187]. It had received much attention during its initial use as a coating material to

690 improve the osseointegration ability of implants. However, the first generation of HA-

691 coated implants manufactured with plasma spray technology was withdrawn due to the

692 unacceptable marginal bone loss found in clinical studies [188]. With the advancement

693 of processing technology, several new HA-based coatings for bone regeneration have

694 been developed in recent years. For example, in a recent study conducted by Lu et al.,

695 a nanostructured HA coating was prepared on the titanium surface via electrochemical

696 deposition, in vitro study revealed that osteogenic differentiation of MC3T3 cells on

697 nanostructured HA coatings was relatively more active compared to HA coatings

698 prepared by plasma spray technique without nanotopography. Subsequent in vivo study

699 further confirmed the results in vitro [189]. In an in vitro study conducted by Uddin et

700 al., to enhance corrosion resistance and immune response of Mg alloys implants, deep

701 ball burnishing was first performed to improved surface integrity of Mg alloys implants,

702 then HA coating was manufactured on the magnesium alloy substrate surface via an

703 electrochemical process. In vitro results found that the burnish + HA coating

704 significantly improved the corrosion resistance of the surface when compared with

705 unmodified samples. Besides, the expression level of anti-inflammatory cytokines on

706 the HA coated surface was significantly increased as compared to the uncoated surface
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707 [171], suggesting a favorable immunomodulatory property of this HA coating. A

708 microporous titanium surface that was coated with nano-HA produced by microarc

Journal of Materials Chemistry B Accepted Manuscript

709 oxidization (MAO) and steam-hydrothermal treatment (SHT) techniques was

710 developed by Bai et al. to investigate its capability of osseointegration. By changing

711 the treatment time of SHT, the nano-HA structure can be transformed from

712 nanoparticles to nanorods, in vitro results suggested that HA nanoparticles were able to

713 induce the formation of an immune microenvironment more conducive to osteogenesis

714 and angiogenesis than HA nanorods, which was further supported by the results of

715 subsequent in vivo study [172] (Figure 12). Similarly, another study by Wang et al. also

716 revealed that HA nanoparticles that were also prepared through MAO and SHT

717 promoted M2 polarization of macrophages while stimulating osteogenesis and

718 angiogenesis, and accelerated the process of osseointegration in an in vivo assay [173].

719 These results suggest that HA coatings may be a potential alternative to regulate the

720 immune environment to make it more favorable for bone regeneration. However, Long-

721 term clinical studies are needed to further confirm its safety and efficacy.
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Journal of Materials Chemistry B Accepted Manuscript

722
723 Figure 12. Effect of surface coating on osteoimmunomodulation. (A) Schematic of influence of

724 different surface coatings on immunomodulation and subsequent osseointegration. HA

725 nanoparticles were able to induce an immune environment more conducive to osteogenesis and

726 angiogenesis than HA nanorods. Reprinted with permission [172]. Copyright 2018, Elsevier. (B)

727 Schematic representation of osteoimmunomodulatory effects of implants coated with IL-4/graphene

728 oxide (GO). IL-4/GO-coated titanium surfaces promoted M2 polarization of macrophages and bone

729 regeneration. Reprinted with permission [182]. Copyright 2020, American Chemical Society. (C)

730 Schematic of effect of high Sr-doped coatings on bone regeneration under OS conditions. High Sr-

731 doped coatings could effectively induce the osteogenic differentiation of MC3T3-E1 cells and the

732 M2 polarization of RAW264.7 cells by activating catalase/superoxide dismutase and reducing

733 reactive oxygen species, thus promoting bone regeneration. (D) Fluorescent images of
 Journal of Materials Chemistry B Page 48 of 71

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734 Arg/CD68/DAPI after implantation for 7 days. The fluorescence intensity of Arg (M2DOI:
marker)
10.1039/D3TB00727H

735 increased with the addition of Sr-doped coatings. （C and D）Adapted from ref. [180].

Journal of Materials Chemistry B Accepted Manuscript

736 6.2 Bioactive glass coatings

737 Bioactive glass (BAG) was once presented and claimed to be capable of eliciting

738 chemical response at the material surface, thus forming a bond between the host and

739 the material [190]. Latter, clinical application of BG as coating of orthopedic or dental

740 implants was proposed because of its reported good osteoconductivity, osteoinductivity,

741 and biocompatibility [191, 192]. However, the inherent low fracture toughness and high

742 brittleness of BAG have limited their clinical application in osseointegration [193].

743 Until today, no BAG coating for orthopedic or dental implants is approved for clinical

744 application, but some of the emerging studies seem promising. For instance, in a study

745 conducted by Chen and colleagues, a collagen membrane that was coated with

746 nanometer-sized bioactive glass Ca2ZnSi2O7 via pulsed laser deposition was

747 developed to investigate its role in osteoimmunomodulation. Both in vitro and in vivo

748 studies revealed that BAG coatings upregulated the M1 polarrization of macrophages,

749 meanwhile enhanced osteogenic differentiation of BMSCs associated with BAG

750 coatings was also detected [174]. In another study by Zhang et al., a β -tricalcium

751 phosphate (TCP) scaffold coated with mesoporous BAG nanolayer was developed

752 through 3D printing and spin coating to improve the capabilty of osseointergration of

753 pure TCP scaffold. Mechenical tests showed that the compressive strength of the

754 mesoporous BAG nanolayers modified TCP scaffolds is about 11 MPa, which is in the

755 top range for cancellous bone. In vitro study revealed that upregulated osteogenesis and
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756 angiogenesis were noted on mesoporous BAG nanolayers modified TCP scaffolds as

757 compared to pure TCP scaffolds and traditional BAG modified TCP scafolds. Similarly,

Journal of Materials Chemistry B Accepted Manuscript

758 the mesoporous BAG nanolayer modified TCP scaffolds exhibited the best

759 osseointergration in subsequent in vivo study [194]. Although several studies have

760 confirmed that BAG coating can improve the osseointegration ability of implants, there

761 is still a lack of experiments to verify its long-term performance in vivo. However, the

762 application prospect of nanostructure BAG as an implant coating cannot be denied.

763 6.3 Extracellular matrix coatings

764 The extracellular matrix (ECM) is a kind of naturally derived biomaterials with

765 good biocompatibility and cell reactivity, which is considered as a potential alternative

766 scaffold material in bone tissue engineering [195, 196]. In an in vivo study, ECM

767 coating screws were implanted into the vertebraes of young sheep to evaluate the ability

768 of osseointegration of ECN coatings. Enhanced bone formation was observed around

769 the surface of the ECM coating screws as compared to the pure screws, indicating the

770 possibility of ECM as a coating material [197]. In an in vivo study conducted by

771 Rahmati et al., enamel matrix derivates (EMD) were coated on the titanium surfaces

772 via electrochemical cathodic polarization method. Then the tibia defect models of

773 rabbits were utilized to evaluate the osteoimmunomodulatory effects of the EMD

774 modified implants. In vivo study revealed that EMD modified implants significantly

775 increased ALP activity and reduced the expression of pro-inflammatory cytokines,

776 suggesting an osteoimmunomodulatory effect of EMD [176]. In another study, a

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777 biomimetic glycopeptide hydrogel was designed by Wang et al. to mimic the

778 glycoprotein composition and the fibrous structure of a natural extracellular matrix and

Journal of Materials Chemistry B Accepted Manuscript

779 coated on a PCL/nHA scaffold for bone regeneration. In vitro study revealed that

780 enhanced proliferation and osteogenic differentiation of BMSCs was found on coated

781 surface, which was further promoted by the M2 macrophage polarization induced by

782 the glycopeptide hydrogel, in vivo study further confirmed the enhanced

783 osteoimmunomodulatory effects of this hybrid scaffold developed here [170] (Figure

784 13). Existing studies suggest that ECM coating can regulate the immune

785 microenvironment to accelerate osteogenesis, but more in vivo studies are needed to

786 confirm these hypotheses.

787
788 Figure 13. Effect of extracellular matrix coating on osteoimmunomodulation. (A) GM-RADA16

789 hydrogel (GRgel) was combined with PCL/nHA to form a new scaffold (PH@GRgel). (B-C) Bone

790 healing is achieved by GR-mediated immune-modulation and RADA16-induced osteogenesis. (D)

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791 Fluorescent images of macrophages polarization markers at the new bone area, with DOI:
a higher
10.1039/D3TB00727H

792 percentage of M2 cells observed at the regenerated tissue treated by PH@GRgel. Reprinted with

Journal of Materials Chemistry B Accepted Manuscript

793 permission [170]. Copyright 2022, Elsevier.

794 6.4 Metal ions coatings

795 Metal ions can inhibit inflammatory cytokines secreted by macrophages and

796 promote bone formation [198, 199]. Several researchers have attempted to design

797 immunomodulatory implants by coating metal ions on the surfaces of the implants. For

798 instance, a titanium surface coated with tannic acid and magnesium ions was prepared

799 by He et al. to investigate its osteoimmunomodulatory properties. In vitro results

800 showed that tannic acid and magnesium ions could synergistically stimulate the

801 selective polarization of macrophages toward the anti-inflammatory phenotype, thereby

802 promoting osteogenic differentiation of BMSCs. In vivo study also revealed that the

803 coatings developed hear could inhibit the host response [168]. In another study, a

804 magnesium ion-incorporated TiO nanotube array (MgN) was coated on a titanium

805 substrate via anodization and hydrothermal techniques, both in vitro and in vivo studies

806 demonstrated that the MgN coating possessed immunomodulatory properties and was

807 capable of facilitating osteogenesis [177]. In a study by Wu et al., MgSiO3 was

808 developed as a coating material to improve the osseointegration of engineered implants,

809 in vitro study found that the MgSiO3 coatings induced an immunomodulation more

810 favorable for osseointegration as compared to the HA coatings, manifested by increased

811 M2 polarization of macrophages, enhanced osteogenesis, and inhibition of

812 osteoclastogenesis. Moreover, subsequent in vivo study further confirmed that more
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813 osseointegration were observer on the MgSiO3 coatings than the HA coatings [178].

814 In addition to magnesium ion, other metal ions have also been demonstrated to possess

Journal of Materials Chemistry B Accepted Manuscript

815 immunomodulatory properties. For instance, Lu et al. constructed a strontium-

816 incorporated titanium using phase-transited lysozyme technique. In vitro results

817 showed that Sr-loaded surface could induce the M2 polarization of macrophages and

818 thus stimulated osteogenic differentiation of BMSCs. In vivo study using the femur

819 defect models of rats revealed that Sr-loaded surfaces could alleviate inflammatory

820 response while increasing bone formation [179]. Another study conducted by Shen et

821 al. found that the high Sr-doped coatings could effectively induce the osteogenic

822 differentiation of MC3T3-E1 cells and the M2 polarization of RAW264.7 cells in vitro

823 by activating catalase/superoxide dismutase and reducing reactive oxygen species, thus

824 promoting bone regeneration. In vivo study was also performed and found that the high

825 Sr-doped samples exhibited favorable effects on osteoimmunomodulation under the

826 oxidative stress microenvironment [180] (Figure 12). In Another study, Liu et al.

827 fabricated zinc ion coatings on the sulfonated polyetheretherketone (SPEEK) surfaces

828 via magnetron sputtering technique. Both in vitro and in vivo studies revealed that Zn-

829 coated SPEEK could modulate macrophages to polarize into an anti-inflammatory

830 phenotype and promote osteogenic differentiation of BMSCs subsequently, suggesting

831 that zinc is a promising option for endowing biomaterials with

832 osteoimmunomodulatory properties [200]. These results indicate that metal ion

833 coatings can endow the implant with immunomodulatory properties favoring
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834 osteogenesis, and therefore have great potential as a coating material to promote

835 osseointegration.

Journal of Materials Chemistry B Accepted Manuscript

836 6.5 Anti-inflammatory cytokines loaded coatings

837 A previous study documented that the addition of anti-inflammatory cytokines to

838 implants can stimulate the M2 macrophage polarization [68, 201, 202]. Along with the

839 in-depth research on immune regulation during the process of bone union, some

840 researchers have attempted to incorporate anti-inflammatory cytokines into coatings to

841 achieve an early burst release of cytokines, thereby switching macrophages toward the

842 M2 phenotype in time to facilitate bone regeneration. For instance, IL-4 and RGD

843 peptide were employed by Li et al. to construct a coating on the surfaces of TiO2

844 nanotubes for the purpose of regulating the immune response and promoting

845 osteogenesis. Cell co-culture models were used to investigate response of macrophages

846 on various surfaces and the modulation of MSCs osteogenic differentiation. In vitro

847 stuies confirmed that this coating could not only switch macrophages toward an anti-

848 inflammatory phenotype, but also promote osteogenic differentiation of MSCs by

849 activating the BMP/SMAD/RUNX2 signaling pathways [181] (Figure 14). In another

850 study, IL-4/graphene oxide (GO) coatings were constructed on titanium substrates to

851 improve the immunomodulatory properties of the scaffolds. In vitro study found that

852 the coated scaffolds could promote the M2 macrophage polarization and the osteogenic

853 differentiation of BMSCs. Moreover, in vivo studies revealed that osseointegration at

854 the interface of the coated implant was enhanced [182] (Figure 12). A study by Xie et
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855 al. designed a PEEK implant coated with a programed surface to achieve sequential

856 release of IL-10 and dexamethasone. In vitro study revealed that a burst release of IL-

Journal of Materials Chemistry B Accepted Manuscript

857 10 and sustained slow delivery of dexamethasone induced an inflammatory

858 microenvirment at first, followed by M2 polarization of macrophages, the continuous

859 release of dexamethasone combined with the anti-inflammatory microenvironment

860 promoted bone regeneration synergistically. Similarly, significantly increased

861 osseointegration on the modified surface was also observed in vivo [203]. All these

862 studies reveal that the introduction of anti-inflammatory cytokines to the implant

863 surfaces may be a possible way to regulate the immune environment favoring bone

864 regeneration. Furthermore, the appropriate timing of immune modulation is equally

865 important and requires further investigation.

866
867 Figure 14. Schematic of the effect of anti-inflammatory cytokines loaded coating on

868 osteoimmunomodulation. L-4 and RGD can work synergistically and generate a better osteo-

869 immune microenvironment, which is more favorable for early osteogenesis. Reprinted with

870 permission [181]. Copyright 2020, Elsevier.

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871 6.6 Drug-loaded coatings

872 To date, several small molecule drugs have been demonstrated to possess an

Journal of Materials Chemistry B Accepted Manuscript

873 osteoimmunomodulatory function during the process of osteogenesis [167, 204]. The

874 development of material science has made it possible to cover these drugs on the

875 surfaces of the implants. For instance, a drug-loaded coating material with

876 inflammation-responsive release function was synthesized by He et al. using

877 indomethacin and tannic acid. In the present study, indomethacin and tannic acid

878 exhibited a synergistic immunomodulatory effect, manifested as the downregulated

879 pro-inflammatory cytokines and the upregulated anti-inflammatory cytokines, which

880 ultimately promoted osteogenic differentiation of BMSCs in vitro. Meanwhile,

881 significantly increased osseointegration was observed on this novel coating in vivo

882 [167]. In another study, the effects of aspirin-loaded coatings on osteogenesis and

883 immunomodulation around implants were studied by Zhang et al., Aspirin-loaded

884 coatings were decorated on titanium discs through electrostatic interactions. Increased

885 M2 polarization of macrophages and enhanced osteogenic differentiation of BMSCs

886 were shown on the aspirin modified disc as compared to the pure disc, subsequent in

887 vivo results mirrored the findings in vitro. [184]. Recently, Zheng et al. used IL-4,

888 alendronate and nanohydroxyapatite to synthesize a hybrid coating capable of

889 programmed release to improve the osseointegration ability of implants. Both in vitro

890 and in vivo studies suggested that the initial burst of IL-4 in the early stage significantly

891 stimulated the M2 polarization of macrophages, which promoted bone regeneration

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892 subsequently. Over the next few weeks, the sustained release of alendronate and Ca2+

893 inhibited osteoclastogenesis while promoting osteogenesis, effectively improved the

Journal of Materials Chemistry B Accepted Manuscript

894 osseointegration capacity of the implants developed here [183] (Figure 15). Taken

895 together, these results suggested that drug-loaded coatings may endow implants with

896 favorable immunomodulatory properties and provide strategies for the surface

897 modification of engineering implants. To date, however, few studies have investigated

898 the immunomodulatory properties of drug-loaded coatings, and little consensus exists

899 for the determination of drug components or concentrations. These problems need to

900 be addressed by further researches, especially in vivo experiments.

901
902 Figure 15. Schematic of the effect of drug-loaded coating on osteoimmunomodulation. A

903 programmed release of IL-4, ALN and Ca2+ can work cooperatively to match the dynamic process

904 of bone regeneration. Adapted from ref. [183].

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905 6.7 Multicomponent coatings

906 It is worth mentioning that some studies have attempted to achieve a multifaceted

Journal of Materials Chemistry B Accepted Manuscript

907 synergistic improvement of the osseointegration capability of implants by adding

908 coating components. For example, in our aforementioned study by Zheng et al. , IL-4,

909 alendronate, and nano-hydroxyapatite were all involved in the formation of this hybrid

910 coating, in which IL-4 was used to modulate the immune response while alendronate

911 and calcium ions were used to inhibit osteolysis and promote osteogenesis, thereby

912 improving the osseointegration of the implant [183]. In another study, He and

913 colleagues selected tannic acid (TA) and Mg2+ to build a multicomponent coating based

914 on metal-phenolic chemistry. Among them, Mg2+ was demonstrated to be able to

915 regulate the M2 polarization of macrophages and promote osteogenesis, TA could

916 scavenge excess reactive oxygen species (ROS). This TA/Mg2+ coating integrated the

917 above functions, and showed excellent immune regulation ability in subsequent in vivo

918 experiments [168]. It is undeniable that multicomponent coatings provide a new

919 direction for the development of multifunctional biomaterials, but more rigorous in vivo

920 experiments are needed to verify their safety and effectiveness, as well as the reasonable

921 proportion of each component.

922 7. Conclusions and Future Perspectives

923 Immune cells play vital roles in regulating bone regeneration. Immune response is

924 inevitable to any biomaterial. Traditional design principles tend to minimize the

925 immune response of biomaterials. However, unsatisfactory results were described in

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926 previous studies. As the emergence of a new concept called osteoimmunology, the

927 intimate relationship between bone and immunity has been thoroughly studied, and

Journal of Materials Chemistry B Accepted Manuscript

928 cumulative evidence has demonstrated that a proper immune environment is favorable

929 for bone regeneration, which facilitates the transformation of design methods from

930 immune evasive to immunomodulation. Commonly, after the implantation of

931 biomaterials, blood clots and proteins are immediately absorbed on the biomaterial

932 surfaces, further determining the cascade of events driven by the immune system, which

933 ultimately affects the results of bone regeneration. By tuning surface properties,

934 biomaterials can precisely manipulate protein adsorption and the subsequent immune

935 response, resulting in favorable bone regeneration. Hence, various surface properties,

936 including topography, roughness, porosity, pore size, wettability, surface charge,

937 functional groups, and surface coatings, have been investigated and demonstrated to

938 participate in immunoregulatory processes. The highly tunable characteristics and

939 immunomodulatory effects make surface modification a viable solution for application

940 in bone tissue engineering. We noted that among the immune cells involved in

941 osteoimmunomodulation, macrophages were studied most due to their capability of

942 switching their phenotypes in response to different inducers. The appropriate

943 conversion of macrophage phenotypes at the right time is essential for successful bone

944 regeneration, while no consensus has yet been reached regarding the appropriate timing

945 and pattern of macrophage conversion. Moreover, we noted that other immune cells,

946 such as T lymphocytes and B lymphocytes, have received less attention than
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947 macrophages. Future work should focus more on these cells. After all,

948 osteoimmunomodulation is a process in which a variety of immune cells participate. In

Journal of Materials Chemistry B Accepted Manuscript

949 addition, with regard to the same surface modification method, different parameter

950 designs will also lead to differences in the immunomodulatory effect of biomaterials.

951 How to define the optimal surface parameters also needs to be further clarified through

952 rigorous experiments. Whether the final surface modification of biomaterials can meet

953 our needs still needs to be proved by clinical studies. In summary, surface modification

954 can be a viable solution for bone regeneration by precisely manipulating the immune

955 cell response, providing new directions for the management of bone defects.

956

957 Acknowledgments:

958 The authors thank Prof. Yujiang Fan and Prof. Xingdong Zhang for their help in this

959 study. This work was supported by the National Natural Science Foundation of China

960 (82202705), the Project of the Science and Technology Department of Sichuan

961 Province (Grant No. 2022YFS0099, No. 2023NSFSC1738, No. 2023YFS0162), the

962 Sino-German Center for Research Promotion (GZ1219). In addition, some elements

963 in the figures are created through BioRender.

964

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