Exosome-integrated titanium oxide nanotubes for targeted bone regeneration

Accepted Manuscript Full length article Exosome-integrated titanium oxide nanotubes for targeted bone regeneration Fei Wei, Mengting Li, Ross Crawford, Yinghong Zhou, Yin Xiao PII: DOI: Reference:

S1742-7061(19)30017-0 https://doi.org/10.1016/j.actbio.2019.01.006 ACTBIO 5862

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

22 September 2018 2 January 2019 6 January 2019

Please cite this article as: Wei, F., Li, M., Crawford, R., Zhou, Y., Xiao, Y., Exosome-integrated titanium oxide nanotubes for targeted bone regeneration, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio. 2019.01.006

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Exosome-integrated titanium oxide nanotubes for targeted bone regeneration

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Fei Wei1, 2#, Mengting Li 1, 2, Ross Crawford 1, 2, Yinghong Zhou 1, 2, 3*#, and Yin Xiao

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1, 2, 3*

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1

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Technology, Brisbane, 60 Musk Avenue, Kelvin Grove, Brisbane, Queensland 4059,

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Australia

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2

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(ACCTERM), Queensland University of Technology, Brisbane, 60 Musk Avenue,

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Kelvin Grove, Brisbane, Queensland 4059, Australia

The Institute of Health and Biomedical Innovation, Queensland University of

The Australia-China Centre for Tissue Engineering and Regenerative Medicine

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3

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Stomatology Hospital of Guangzhou Medical University, Guangzhou 51050, China

Key Laboratory of Oral Medicine, Guangzhou Institute of Oral Disease,

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#Fei

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first authors

Wei and Yinghong Zhou contributed equally to this work and are considered co-

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*Correspondence authors:

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Yinghong Zhou

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Tel: 61- 7 3138 6269

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Fax: 61-73138 6030

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E-mail: [email protected]

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22

Yin Xiao

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Tel: 61-731386240

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Fax: 61-73138 6030

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E-mail: [email protected]

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Running title: Exosome-integrated titanium nanotubes for bone regeneration

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Abstract

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Exosomes are extracellular nanovesicles that play an important role in cellular

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communication. The modulatory effects of bone morphogenetic protein 2 (BMP2) on

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macrophages have encouraged the functionalization of scaffolds through the

45

integration of the exosomes from the BMP2-stimulated macrophages to avoid ectopic

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bone formation and reduce adverse effects. To determine the functionality of

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exosomal nanocarriers from macrophages after BMP2 stimulation, we isolated the

48

exosomes from Dulbecco’s modified Eagle’s medium (DMEM)- or BMP2-stimulated

49

macrophages

50

characterization of the exosomes derived from DMEM- or BMP2-treated

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macrophages revealed no significant differences, and the bone marrow-derived

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mesenchymal stromal cells showed similar cellular uptake patterns for both exosomes.

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In vitro study using BMP2/macrophage-derived exosomes indicated their beneficial

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effects on osteogenic differentiation. To improve the bio-functionality for titanium

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implants, BMP2/macrophage-derived exosomes were used to modify titanium

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nanotube implants to favor osteogenesis. The incorporation of BMP2/macrophage-

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derived exosomes dramatically increased the expression of early osteoblastic

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differentiation markers, alkaline phosphatase (ALP) and BMP2, indicative of the pro-

59

osteogenic role of the titanium nanotubes incorporated with BMP2/macrophage-

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derived exosomes. The titanium nanotubes functionalized with BMP2/macrophage-

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derived exosomes activated autophagy during osteogenic differentiation. In

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conclusion, the exosome-integrated titanium nanotube may serve as an emerging

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functional material for bone regeneration.

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Keywords: BMP2; macrophages; exosome; titanium nanotubes; osteogenesis.

and

evaluated

their

effects

on

osteogenesis.

Morphological

65

1. Introduction

66

Bone fractures are very common and affect 2% of the population per annum,

67

especially in countries with increased obesity and poor levels of physical activity [1,

68

2]. The cost of bone-related diseases will continue to rise with the aging of population,

69

causing a huge annual socioeconomic burden worldwide [3]. In spite of the inherent

70

regenerative ability of bones, many patients display delayed or compromised bone

71

healing and need therapeutic intervention [4].

72

Titanium and titanium-based alloys are one of the most widely used scaffolds for

73

clinical implantation, owing to their excellent biocompatibility and mechanical

74

properties [5]. Being relatively bio-inert, numerous technologies, including physical

75

and chemical treatments, have been applied to modify the titanium surface [6]. Such

76

surface modification techniques and micro-texture changes on titanium scaffold, offer

77

different microenvironments to facilitate fundamental interactions between tissues and

78

implants [7]. Microtopographical growth of nanotubes on titanium substrate has

79

gained attention, owing to the significant improvement in the in vitro proliferation and

80

differentiation of mesenchymal stromal cells (MSCs) [8, 9]. Previous studies have

81

described the application of titanium nanotubes as a unique three-dimensional

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reservoir for osteogenic agents [10]. Among the osteoinductive molecules discovered

83

so far, bone morphogenetic protein 2 (BMP2) is the most widely used growth factor

84

for bone regeneration [11]. Several implantable carriers such as absorbable collagen

85

sponges [12], polylactic-co-glycolic acid (PLGA) microspheres [13], and titanium

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nanotube-based carriers [10] have been tested as drug delivery systems for BMP2 in

87

vitro and in vivo [14]. Titanium nanotubes alone or in combination with other

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materials have been successfully exploited as BMP2 reservoir for MSC differentiation

89

[15]. For instance, gelatin/chitosan-coated titanium nanotubes/BMP2 structure could

90

promote the osteoblastic differentiation of MSCs [15]. A more recent study attempted

91

to enhance bone regeneration in rabbits using titanium nanotube array through the

92

integration of BMP2 [16]. However, several side effects such as ectopic bone

93

formation, inflammation, bone resorption, and hematoma have been reported in the

94

clinical use of BMP2 [17]. In addition, high dosage of BMP2 is needed owing to the

95

poor retention rate of some BMP2 carriers, thereby comprising the safety application

96

of BMP2 [18].

97

Exosomes are naturally secreted vesicles from cells that play an important role in

98

intercellular communication. Previous studies have shown that exosomes are

99

important nanocarriers for transferring proteins, lipids, and genetic information from

100

parent cells to recipient cells [19]. The role of MSC-derived exosomes has been

101

previously evaluated, owing to the regulatory effects of MSCs on collagen-induced

102

inflammatory arthritis models [20]. In addition, the beneficial effects of the MSC-

103

derived exosomes have also been observed in various animal models such as those of

104

liver fibrosis and carbon tetrachloride-induced acute liver injury [21]. The effects of

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immune cell-derived exosomes on immune response and inflammation have been

106

previously studied [22]. Macrophage-derived exosomes are one of the most abundant

107

sources of microvesicle populations in the peripheral blood [23]. As an essential

108

component of the innate and adaptive immunity, activated macrophages are important

109

regulators in inflammation, host defense, and tissue regeneration [24]. Macrophages

110

may be classically activated to M1 or M2 phenotypic profile depending on the

111

environmental cues [25]. For instance, M1-polarized macrophages induced by

112

lipopolysaccharide (LPS) stimulation increase exosome secretion, which in turn, plays

113

an important regulatory role in inflammation [26]. Activation of macrophages

114

following BMP2 or BMP7 treatment has also been previously recorded in vitro and in

115

vivo [27-29].

116

Although these studies show the promising applications of exosomes in regenerative

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medicine, the knowledge of the bone regenerative potential of the macrophage-

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derived exosomes is limited. Therefore, here we investigated the regulatory role of

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BMP2/macrophage-derived exosomes in the osteogenic differentiation of MSCs. The

120

regenerative potential of BMP2 has often been accompanied with certain adverse

121

events and complications. Our study focused on the fabrication of exosome-based

122

titanium nanotubes to enhance the osteogenic potential of BMP2 via natural

123

nanocarriers. The results presented herein may provide new insights into the

124

application of titanium nanotube-based materials for the safe use of BMP2.

125 126

2. Materials and Methods

127

2.1 Cell culture, stimulation, and exosome isolation

128

A murine-derived macrophage cell line, RAW 264.7 (ATCC® TIB-71™), and human

129

bone marrow mesenchymal stromal cells (hBMSCs) were used in this study. RAW

130

264.7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM;

131

Gibco®, Life Technologies Pty Ltd., Australia) supplemented with 10% heat-

132

inactivated fetal bovine serum (FBS; In Vitro Technologies, Australia), and 1% (v/v)

133

penicillin/streptomycin (Gibco®, Life Technologies Pty Ltd., Australia),

134

as previously described [30]. The handling of the human samples was approved by the

135

Office of Research Ethics and Integrity of Queensland University of Technology

136

(QUT). Bone marrow samples were collected from six patients undergoing elective

137

knee replacement surgery at the Prince Charles Hospital after obtaining a full

138

informed consent. Mononuclear cells were acquired from the human bone marrow

139

with density gradient centrifugation using Lymphoprep (Axis-Shield PoC AS, Oslo,

140

Norway) according to the manufacturer’s instructions. Cells were maintained in

141

DMEM supplemented with 10% FBS and 1% (v/v) penicillin/streptomycin in a

142

humidified incubator containing 5% CO2 at 37°C. The nonadherent hematopoietic

143

cells were removed via changing the medium. MSCs from different donors were

144

initially separately cultured and pooled at passage 2, as previously described [31].

145

Osteogenic medium was prepared as previously described [32]. In brief, hBMSCs

146

were stimulated with 10% DMEM supplemented with osteogenic components (2 mM

147

β-glycerophosphate, 100 μM l-ascorbic acid 2-phosphate, and 10 nM dexamethasone;

148

Sigma-Aldrich, NSW, Australia).

149

RAW 264.7 cells were stimulated with DMEM (Ctr-exo) or 100 ng/mL of BMP2

150

(355-BM-100, R&D System Inc.) for 12 h. After washing thrice with phosphate-

151

buffered saline, the cells were cultured with 10 mL of serum-free DMEM at 37°C for

152

12 h prior to the collection of the conditioned medium (CM). CM was centrifuged at

153

300 g and 4°C for 10 min and filtered through 0.22 µm filters to remove

154

contaminated apoptotic bodies, microvesicles, and cell debris. Total exosomes were

155

isolated from CM based on protocols from a previous study [33]. In brief, the

156

supernatants were spun at 100,000 g and 4°C for 90 min to pellet exosomes. After

157

carefully removing the supernatant, exosomes were resuspended in 2 mL of ice-cold

158

phosphate-buffered saline (PBS). Samples were spun at 100,000 g and 4°C for 90

159

min, and the resulting exosome pellets were resuspended in 100 µL of PBS and stored

160

at −80°C immediately until further analysis.

161

2.2 Transmission electron microscopy (TEM)

162

TEM was used to verify the presence of exosomes in the purified samples. Briefly, 5

163

μL of the extracted exosomes were placed on to carbon/formvar-coated Cu TEM grids

164

(Lot #090913) for 10 min. After staining with 1% uranyl acetate for 20 s, the grids

165

were washed twice in deionized water, gently blotted on Whatman filter paper, and

166

air-dried. The exosomes were imaged by TEM (JEM-1400, JOEL, Japan) at 80 kV.

167

2.3 Exosome labeling with PKH67

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Exosomal uptake by hBMSCs was monitored with PKH67 Green Fluorescent Cell

169

Linker Mini Kit (Lot #MINI67-1KT, Sigma-Aldrich, NSW, Australia) according to

170

the manufacturer’s instructions. Briefly, 10 μL of Ctr-exo or BMP2-exo were mixed

171

with 240 μL of Diluent C fluid (Part A). After adding 1 μL of PKH67 ethanolic dye

172

solution to 250 μL of the Diluent C fluid (Part B), 250 μL of exosome suspension

173

(Part A) was mixed with 250 μL of Dye solution (Part B) for 3 min at room

174

temperature (25°C) . The staining process was terminated with the addition of 1 mL

175

1% FBS-containing DMEM (exosome-free) for 1 min. The labeled exosomes were

176

incubated with hBMSCs for 12 h at 37°C. Samples were fixed with 4%

177

paraformaldehyde (PFA, Lot #P6148, Sigma-Aldrich, Australia) and mounted on

178

glass slides with ProLong® anti-fade reagents (Life Technologies Pty Ltd., Australia).

179

Images were captured using a confocal laser scanning microscope (Nikon Air

180

Confocal, Australia).

181

2.4 Real-time quantitative reverse-transcription polymerase chain reaction

182

(qRT-PCR)

183

hBMSCs were cultured in the osteogenic medium supplemented with an equal

184

amount of Ctr-exo or BMP2-exo for 3 days. Total RNA was extracted using TRIzol®

185

reagent (Lot #15596-018, AmbionTM, Life Technologies Pty Ltd., Australia)

186

according to the manufacturer’s instructions. Reverse transcription was performed

187

using DyNAmo™ cDNA Synthesis Kit (Finnzymes, Genesearch Pty Ltd., Australia).

188

qRT-PCR was performed using QuantStudioTM Real-Time PCR System instrument

189

(Applied Biosystems, USA) according to a two-step PCR protocol (95°C for 2 min,

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45 cycles of 5 s at 95°C, 10 s at 60°C, and 15 s at 72°C). Primers were designed and

191

purchased from Sigma-Aldrich, Australia. All primer sequences are shown in Table

192

S1. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an

193

endogenous control to normalize the differences in the amount of total RNA in each

194

sample. Samples were evaluated and analyzed in triplicates using the comparative Ct

195

(2−ΔΔCT) method [34, 35].

196

2.5 Encapsulation of exosomes into titanium nanotubes

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Titanium nanotubes were generated by electrochemical anodization, as previously

198

described [36]. Briefly, titanium disks were incubated with 5% ethylene glycol

199

(Sigma-Aldrich, Australia) and 0.3% ammonium fluoride solution (Sigma-Aldrich,

200

Australia) at 30 V for 1 h before thorough rinsing with deionized water.

201

Poly(dopamine) (Lot #H8502, Sigma-Aldrich, Australia) coating was performed

202

based on the published protocol [37]. For exosome coating, equal amounts of

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exosomes were incubated with titanium nanotubes for 1 h at room temperature,

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followed by rinsing thrice with sterile PBS. For the determination of exosome

205

integration into titanium nanotubes, equal amounts of exosomes (Ctr-exo or BMP2-

206

exo) were pre-labeled with PKH67 using PKH67 Green Fluorescent Cell Linker Mini

207

Kit. The labeled exosomes were incubated with titanium nanotubes for 1 h at room

208

temperature and then rinsed thrice with sterile PBS. Encapsulation of exosomes into

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titanium nanotubes was visualized using a confocal laser scanning microscope (Nikon

210

Air Confocal, Australia).

211

2.6 Release study

212

The release of exosomes from titanium nanotubes was monitored with an uptake

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assay. Briefly, equal amounts of exosomes (Ctr-exo or BMP2-exo) were pre-labeled

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with PKH67. The labeled exosomes were incubated with titanium nanotubes for 1 h at

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room temperature and then rinsed thrice with sterile PBS. Ctr-exo/NT or BMP2/exo-

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NT were incubated with hBMSCs in the co-culture system, and samples were

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collected at 1, 4, 8, and 12 post-incubation. hBMSCs were fixed with 4% PFA and the

218

images were captured under a confocal laser scanning microscope (Nikon Air

219

Confocal, Australia).

220

2.7 Western blot analysis

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hBMSCs were cultured on Ctr-exo/NT or BMP2-exo/NT supplemented in osteogenic

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medium for 3 and 7 days. The medium was changed on day 3 without any additional

223

supplementation of exosomes. The whole cell lysates were collected by adding 200

224

μL radioimmunoprecipitation assay (RIPA) buffer (Lot #R0278, Sigma-Aldrich,

225

Australia) with protease inhibitor (cOmplete, ethylenediaminetetraacetic acid

226

[EDTA]-free

227

04906845001, Roche). For exosomal marker identification, exosome isolations were

228

also lysed with RIPA buffer. Equal amounts of proteins (15 μg) were resolved on

229

10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for 90

230

min. The gels were transferred onto 0.45-μm nylon-supported nitrocellulose

231

membranes and incubated with Odyssey® Blocking Buffer (LI-COR Biotechnology,

232

USA) at room temperature for 1 h. The blots were incubated with appropriate primary

04693132001,

Roche)

and

phosphatase

inhibitor

(PhosSTOP,

233

antibodies overnight at 4°C (α-tubulin, 1:1000, ab4074, Abcam; alkaline phosphatase

234

[ALP], 1:1000, ab108337, Abcam; β-catenin. 1:1000, ab4074, Abcam; BMP2, 1:1000,

235

ab82511, Abcam; autophagy-related protein 5 [ATG5], 1:1000, 12994, Cell Signaling

236

Technology; microtubule-associated protein light chain 3 [LC3]-I/II, 1:1000, 12741,

237

Cell Signaling Technology; Alix, 1:1000, 2171, Cell Signaling Technology; Annexin

238

V, 1:1000, 8555, Cell Signaling Technology). After washing with PBS-Tween

239

20 (0.1%), the blots were incubated with IRDye® 800CW goat anti-rabbit IgG (H+L)

240

or IRDye® 680RD goat anti-mouse IgG (H+L) (1:5000; LI-COR Biotechnology, USA)

241

and washed thrice with PBS-Tween 20 (0.1%). Protein signals were visualized using

242

Odyssey infrared Imaging System (LI-COR Biotechnology, USA). The relative

243

intensity of protein bands compared with α-tubulin was quantified using Image

244

StudioTM

245

download-image-studio-lite-software/) [35].

246

2.8 Immunofluorescence staining and confocal imaging

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hBMSCs were cultured on Ctr-exo/NT or BMP2-exo/NT for 3 and 7 days in

248

osteogenic medium. For ALP staining, cells were fixed with 4% PFA, permeabilized

249

with 0.25% Triton X-100 (Lot #T8787, Sigma-Aldrich, NSW, Australia) for 10 min,

250

and blocked with 4% bovine serum albumin (BSA) for 1 h at room temperature, prior

251

to incubation with rabbit polyclonal antibody against anti-ALP antibody (ab108337,

252

Abcam) overnight at 4°C. Fluorescein isothiocyanate-conjugated goat anti-rabbit IgG

253

(Lot #F6005, Sigma-Aldrich, Australia) was used as a secondary antibody. Actin was

254

stained with Alexa Fluor 594-labeled phalloidin (Lot #A12381, Life Technologies Pty

255

Ltd., Australia). Samples were mounted on glass slides with ProLong® anti-

256

fade reagents (Life Technologies Pty Ltd., Australia). Images were visualized using a

Software

(http://www.licor.com/bio/blog/software-and-accessories/free-

257

confocal laser scanning microscope (Nikon Air Confocal, Australia). The corrected

258

total cell fluorescence (CTCF) was measured using ImageJ according to the following

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equation: CTCF = Integrated density − (Area of selected cell × Mean fluorescence of

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background readings) [38].

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2.9 Alkaline phosphatase assay

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hBMSCs were cultured on Ctr-exo/NT or BMP2-exo/NT for 3 and 7 days in

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osteogenic medium. ALP activity was measured using an alkaline phosphatase assay

264

kit (Colorimetric, ab83369, Abcam) according to the manufacturer’s instruction. Cells

265

were harvested, lysed, and centrifuged, and the supernatants were incubated with p-

266

nitrophenyl phosphate (pNPP) solution in 96-well plates at 25°C for 60 min. The

267

absorbance at 405 nm wavelength was read using BIO-RAD microplate absorbance

268

spectrophotometer.

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2.10 Autophagy detection

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To determine the effect of Ctr-exo/NT and BMP2-exo/NT on the autophagic activity

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of hBMSCs, autophagic vacuoles were detected using Autophagy Detection Kit

272

(ab139484, Abcam) according to the manufacturer's instructions. Briefly, after

273

washing twice with the assay buffer, cells were stained with Dual Detection Reagent

274

for 30 min at 37°C. The cells were washed with the assay buffer and stained with

275

Alexa Fluor 594-labeled phalloidin and 4,6-diamidino-2-phenylindole (DAPI).

276

Samples were mounted on glass slides with ProLong® anti-fade reagents. Images

277

were visualized using a confocal laser scanning microscope (Nikon Air Confocal,

278

Australia).

279

2.11 Cytokine array

280

Cytokine array (Proteome Profiler Human XL cytokine arrays, ARY022, R&D

281

Systems) was performed to study the effect of Ctr-exo/NT and BMP2-exo/NT on the

282

regulation of cytokines produced of hBMSCs. Briefly, membranes spotted with

283

antibodies were incubated with CM at 4°C overnight. After washing, the membranes

284

were incubated with a detection antibody cocktail for 1 h at room temperature and

285

treated with streptavidin-horseradish peroxidase (HRP) solution for 30 min. The

286

signal was visualized using chemiluminescence and exposed to X-ray films.

287

2.12 Statistical analysis

288

All data were expressed as mean ± standard deviations (SD, n = 3). Statistical analysis

289

was performed using GraphPad Prism 7 (Version 7.02) for Windows (GraphPad

290

Software Inc., USA). Statistical differences between groups were determined with

291

one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison-tests.

292

A value of p < 0.05 was considered statistically significant.

293 294

3. Results

295

3.1 Exosome characterization

296

Representative TEM images demonstrate the general morphology of the exosomes

297

isolated from DMEM- or BMP2-treated macrophages (Figure 1A). The isolated

298

exosomes presented a round shape with no significant morphological differences.

299

Representative western blot images revealed the expression of exosome surface

300

markers (Alix and Annexin V) (Figure 1B). To investigate the uptake of exosomes by

301

hBMSCs, the exosomes were labeled with the exosome labeling marker, PKH67, and

302

incubated with hBMSCs. As shown in Figure 2 and Figure S1, the uptake of

303

exosomes was observed in both Ctr-exo and BMP2-exo treated hBMSCs.

304

305

Figure1. Morphological characterization of exosomes derived from control or

306

BMP2 stimulated macrophages. (A) Representative TEM images of exosomes

307

isolated from macrophages stimulated with DMEM or BMP2, respectively. Scale bar:

308

200 nm (a), 100 nm (b), and 90 nm (c). (B) Western blot analysis of the exosome

309

surface markers (Alix and Annexin v).

310 311

Figure2. Characterization of exosome uptake. Representative confocal microscopic

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images indicates the uptake of exosomes by hBMSCs. Low magnification scale bar:

313

50 μm. High magnification scale bar: 25 μm.

314

3.2 Osteogenic activity of hBMSCs stimulated with Ctr-exo or BMP2-exo

315

To investigate the effect of Ctr-exo and BMP2-exo on hBMSC differentiation, equal

316

amounts of exosomes were incubated with hBMSCs for 3 days in osteogenic medium.

317

The expression of osteogenesis-related markers (ALP, osteopontin [OPN], bone

318

sialoprotein [IBSP], run-related transcription factor 2 [Runx2], osteocalcin [OCN],

319

collagen type I [Col-I]) and BMP signaling pathway (BMP2, BMP7, BMP6, Smad1,

320

Smad5, Smad8/9, BMPR1A, BMPRIB, and BMPR2) was detected by qRT-PCR. As

321

shown in Figure 3, the expression of OPN, IBSP, Runx2, OCN, Col-I, BMP2, and

322

BMP7 significantly increased in the cells treated with BMP2-exo, while this effect

323

was absent in the cells treated with Ctr-exo.

324 325

Figure3. Osteogenesis of hBMSCs stimulated by Ctr-exo or BMP2-exo. RNA

326

expression of osteogenesis-related genes (ALP, OPN, IBSP, Runx2, OCN, and Col-I)

327

and BMP signaling pathway (BMP2, BMP7, BMP6, Smad1, Smad5, Smad8/9,

328

BMPR1A, BMPRIB, BMPR2). Data were expressed as the mean ± SD for three

329

independent experiments. *Significant difference (p < 0.05).

330

331

3.3 Characterization of exosome-encapsulated nanotubes and evaluation of

332

exosome uptake by BMSCs

333

As shown in Figure S2, titanium nanotubes were prefabricated before exosome

334

incorporation. To investigate the fabrication of exosomes into titanium nanotubes,

335

exosomes were labeled with PKH67 and incubated with titanium nanotubes for 1 h at

336

room temperature. Integration of Ctr-exo and BMP2-exo on titanium nanotubes was

337

observed in Figure 4, wherein both Ctr-exo/NT and BMP2-exo/NT showed

338

ubiquitous and even distribution of exosomes. Cellular uptake of the exosomes

339

released from Ctr-exo/NT or BMP2-exo/NT was further evaluated. As shown in

340

Figure 5, the uptake of the exosomes released from titanium nanotubes by hBMSCs

341

was evident at 1, 4, 8, and 12 h post-incubation.

342 343

Figure4. Encapsulation of exosomes into titanium nanotubes. Representative

344

confocal microscopic images) of exosome integration into titanium nanotubes. Low

345

magnification scale bar: 200μm. High magnification scale bar: 50μm.

346 347

Figure5. hBMSCsuptake of exosomes released from titanium nanotubes.

348

Representative confocal microscopy images of exosome uptake by hBMSCs released

349

form Ctr-exo/NT (a) or BMP2-exo/NT (b) at indicated time points. Scale bar: 100μm.

350

3.4 Osteogenic activity of hBMSCs stimulated with exosome-encapsulated

351

nanotubes

352

To investigate the effect of the exosome-encapsulated nanotubes on hBMSC

353

differentiation, hBMSCs were stimulated with Ctr-exo/NT or BMP2-exo/NT. Cells

354

were collected at indicated time points and subjected to western blot analysis. As

355

shown in Figure 6A-C, in comparison with the hBMSCs stimulated with Ctr-exo/NT,

356

those cultured with BMP2-exo/NT showed an increase in the expression of early

357

osteoblastic differentiation marker (ALP) and BMP2 after 7 days of stimulation. To

358

evaluate the effect of Ctr-exo/NT or BMP2-exo/NT on hBMSC differentiation, ALP

359

expression was examined with immunofluorescence staining. In comparison with the

360

hBMSCs stimulated with Ctr-exo/NT, those stimulated with BMP2-exo/NP showed a

361

significant increase in the fluorescence intensity of ALP after 7 days of stimulation

362

(Figure 7A-B). A similar trend was observed in the results of ALP activity assay

363

(Figure 7C).

364 365

Figure6. Protein expression of hBMSCs after incubation with exosome-

366

encapsulated nanotubes. (A-C) Expression of early osteoblast differentiation marker

367

(ALP), BMP2, β-catenin, autophagy-related proteins (ATG5, LC3-I, and LC3-II)

368

assessed by Western blot. Asterisk indicates significant difference (p< 0.05). Data

369

were expressed as the mean ± SD for three independent experiments. The protein

370

levels were quantitated by densitometry quantitation normalized to α-Tubulin.

371 372

373 374

Figure7. ALP protein expression of hBMSCs after cultivation with exosome

375

encapsulated nanotubes. (A) Representative confocal microscopic images (n=3) for

376

ALP staining. Scale bar: 50μm. (B) Quantification of ALP fluorescence intensity.

377

Asterisk indicates significant difference (p< 0.05). (C) ALP activity of hBMSCs

378

cultured with Ctr-exo/NT or BMP2-exo/NT. Asterisk indicates significant difference

379

(p< 0.05).

380 381

3.5 Autophagy activation of hBMSCs stimulated with exosome-encapsulated

382

nanotubes

383

Autophagy plays an important role in the self-renewal and differentiation of MSCs

384

[39]. To investigate the effect of the exosome-encapsulated nanotubes on hBMSC

385

differentiation, hBMSCs were cultured with Ctr-exo/NT or BMP2-exo/NT. The

386

conversion of soluble LC3-I to lipidated membrane-bound LC3-II, a marker of the

387

accumulation of autophagic vesicles [40], was examined with western blot analysis

388

and autophagy detection kit. As shown in Figure 6A-C, the accumulation of LC3-II

389

significantly increased in the cells treated with BMP2-exo/NT as compared with those

390

treated with Ctr-exo/NT. Similar results were obtained with the quantification of

391

ATG5, a crucial protein required for autophagy activity [41].

392

To evaluate autophagy activation, autophagic activity was examined with autophagic

393

vacuole staining. As shown in Figure 8, the number of green autophagic vacuole

394

increased in the cells treated with BMP2-exo/NT as compared with those treated with

395

Ctr-exo/NT. These results demonstrate the important regulatory role of BMP2-

396

exo/NT in hBMSC autophagy.

397 398

Figure8. Autophagic activity of hBMSCs stimulated by Ctr-exo/NT or BMP2-

399

exo/NT.Representative confocal microscopic images of hBMSCs stimulated with Ctr-

400

exo or BMP2-exo encapsulated nanotubes for 1, 3 and 7 days under osteogenic

401

medium. Scale bar: 50μm.

402

3.6 BMP2-exo/NT alters the cytokine secretion patterns

403

To evaluate the effect of Ctr-exo/NT and BMP2-exo/NT on the regulation of

404

cytokines, chemokines, and growth factors, a cytokine array was performed using the

405

CM derived from hBMSCs stimulated with Ctr-exo/NT or BMP2-exo/NT (Figure 9).

406

hBMSCs stimulated with BMP2-exo/NT showed several distinct cytokine patterns

407

relative to Ctr-exo/NT control. BMP2-exo/NT induced a significant increase in the

408

production of fibroblast growth factor (FGF)-19, growth/differentiation factor (GDF)-

409

15, interleukin (IL)-17A, IL-19, interferon gamma-induced protein (IP)-10,

410

macrophage inflammatory protein (MIP)-3α, and RANTES. Same cytokine patterns

411

of hBMSCs cultured on titanium nanotubes are shown in Figure S3. These results

412

indicate that BMP2-exo/NT may alter the cytokine secretory levels of hBMSCs.

413 414

Figure9. Cytokines secretion from hBMSCs stimulated with exosomes-

415

encapsulated nanotubes. hBMSCs were incubated with

416

nanotubes or BMP2-exo encapsulated nanotubes under osteogenic differentiation for

Ctr-exo encapsulated-

417

4 days. The conditioned medium collected was measured using proteome profiling

418

human XL cytokine array.

419 420

4. Discussion

421

As one of the most multi-functional organs, the musculoskeletal system controls

422

mineral homeostasis, offers the basic framework for locomotion, and provides other

423

hormones necessary to life [42]. For proper functioning, the musculoskeletal system

424

must cooperate with other systems; the immune system and its critical role in bone

425

remodeling have long been appreciated [43]. This coordinated interaction between the

426

bone and the immune system has led to the generation of a unique interdisciplinary

427

field “osteoimmunology,” which focuses on the molecular understanding of the

428

interplay between the immune and skeletal systems [44]. However, recent

429

investigations have demonstrated that the interaction between the immune system and

430

bone remodeling is more intricate than that previously known [43].

431

Exosomes are nanosized carriers that play an important role in intercellular

432

communication. The role of exosomes in the regeneration of organs and tissues such

433

as heart, lung, and bone has been demonstrated in previous studies [45-47]. The MSC-

434

derived exosomes are known to regulate osteoblast differentiation via an exosomal

435

micro RNA (miRNA) [48]. Exosomes secreted by the immune cells represent

436

important regulators for recipient cells [49]. Stimulation of MSCs with the exosomes

437

isolated from the dendritic cells results in an increase in osteoblastic differentiation

438

[50]. This observation is further supported by an experiment using MSCs that were

439

stimulated with the exosomes derived from LPS-stimulated monocytes in vitro [19].

440

Our experimental results revealed the uptake of the macrophage-derived exosomes by

441

hBMSCs and showed that only BMP2/macrophage-derived exosomes could enhance

442

the osteogenic differentiation of MSCs. The modulatory effects of BMP2 on

443

macrophages have been previously investigated, wherein macrophages were recruited

444

and activated both in vitro and in vivo [27]. Macrophages are one of the major

445

immune cells that regulate the intercellular communication through the release of

446

cytokines and microvesicles, including exosomes [51]. Proteins, lipids, various

447

genetic materials, and low molecular weight metabolites are the main components of

448

exosomes that are transported to recipient cells, wherein they mediate cellular

449

responses to the changing environmental condition [52]. Exosomes derived from

450

BMP2-activated immune cells may, like most exosomes, carry RNA cargo. Recent

451

studies have revealed the important role of some miRNAs in the regulation of bone

452

formation [53]. Therefore, the RNA cargo delivered directly into the hBMSCs may

453

serve as an important cellular regulator for the osteogenic differentiation of hBMSCs.

454

miRNAs isolated from the macrophage exosomes have been implicated in many

455

cellular activities [54, 55]. For instance, phorbol 12-myristate 13-acetate-treated

456

macrophages may increase microvesicle secretion, wherein miR-223 is the most

457

highly expressed and functional miRNA [55]. In addition, the inhibition of miR-223

458

activity by antagomiR may reduce macrophage differentiation, indicating the

459

significant modulatory role of miRNAs in recipient cells [55]. It was recently shown

460

that activated and non-activated macrophages display differential miRNAs profiles,

461

and the expression of miR-530, chr9_22532, and chr16_34840 is abundant in the

462

activated macrophages [56]. Therefore, the exosomes generated by BMP2-stimulated

463

macrophages may be one of the missing links, as our study demonstrates the pro-

464

osteogenesis effects of BMP2/macrophage-derived exosomes on MSC differentiation.

465

However, the regulatory role of the exosomal miRNAs in the modulation of the

466

osteogenic differentiation of hBMSCs needs further investigation. For instance, it

467

would be critical to determine if the specific miRNAs derived from the exosomes of

468

BMP2-stimulated macrophages may induce similar osteogenic effects during tissue

469

repair. Therefore, RNA sequencing is warranted to reveal the modulatory role of the

470

exosomes isolated in this study.

471

We investigated the possibility of integrating the exosomes derived from the BMP2-

472

activated macrophages into titanium nanotubes as important regulatory molecules to

473

enhance osteogenesis. BMP2 is one of the most important osteoinductive molecules.

474

Over the past decades, several carriers have been used for the controlled release of

475

BMP2, such as ceramic-, biopolymer-, or metallic-based delivery system [57]. For

476

instance, beta-tricalcium phosphate (β-TCP) scaffolds incorporated with rhBMP2 can

477

significantly enhance bone formation in a rabbit cranial defect model [58]. Another

478

study using BMP2 titanium nanotubes showed beneficial effects on the proliferation

479

and differentiation of MSCs [10]. Despite the vast advances in delivery systems,

480

growing numbers of adverse effects, especially in patients, have been recorded [18,

481

57]. In comparison with BMP2- or cell-based delivery approaches, exosomes are

482

natural nanosized carriers that may elicit fewer unwanted responses than exogenous

483

osteogenic molecules and allogenic MSCs, thereby serving as a potential therapeutic

484

tool for improved osteogenesis and osteointegration of implants. In addition, different

485

strategies have been proposed to use extracellular vesicles (e.g., exosomes) as natural

486

regulators or drug delivery systems in recent years [52]. In the present study, we

487

focused on the integration of titanium nanotubes with functional exosomes and found

488

that the MSCs showed a sustained uptake of the exosomes released from titanium

489

nanotubes. In comparison with the traditional bio-modification techniques of titanium

490

nanotubes, such as vascular endothelial growth factor (VEGF)/BMP2-enriched

491

titanium implant, and MSC-incorporated titanium implant [59-62], the current

492

strategy for exosomes releasing system may create a favorable osteoenvironment for

493

the differentiation of MSCs.

494

Although the underlying mechanism of action of exosomes in bone regeneration is

495

incompletely understood, the regulatory role of exosomes in autophagic activity

496

presented herein may be essential for the osteogenic differentiation of MSCs.

497

Autophagy is an evolutionarily conserved cellular metabolic process that plays an

498

important role in the maintenance of cellular homeostasis [40]. Autophagy is initiated

499

with the formation of an autophagosome that fuses with lysosomes to form a

500

degradative autophagolysosome, which degrades the engulfed macromolecules and

501

organelles in an acidic environment [40]. It seems that the role of autophagy in

502

osteogenesis and bone remodeling is far more important than that reported. It was

503

shown that the activation of autophagy induced by simvastatin treatment led to a

504

significant increase in the osteogenic differentiation of BMSCs in vitro and

505

osteointegration of oral implants in vivo [63]. In addition, the BMSCs derived from

506

the patients with osteoporosis showed a compromised autophagic activity as

507

compared to those derived from healthy counterparts, indicative of a significant

508

regulatory role of autophagy in bone formation [64]. Depletion of ATG7 led to a

509

significant decrease in osteoclasts and osteoblasts, which in turn, resulted in a

510

remarkable decrease in bone mass compared with the wild-type control [65]. Several

511

studies have reported similar results, highlighting the regulatory role of autophagy in

512

the self-renewal, differentiation, and senescence of MSCs [66, 67]. Therefore, BMP2-

513

exo alone or BMP2-exo/NT may act as important osteogenesis modulators through

514

the autophagy-dependent pathways to support osteogenic differentiation.

515

Bone repair involves four distinct but overlapping stages as follows: initial

516

inflammation response, soft callus phase, hard callus formation, and bone remodeling

517

phase. Soon after the injury, the cytokines produced around the implant areas play an

518

indispensable role in sustenance of MSC recruitment and vascularization [68]. The

519

regulatory role of BMP2-exo/NT in hBMSC differentiation was investigated using a

520

cytokine array. We observed a significant increase in the expression of several

521

candidate cytokines secreted by hBMSCs stimulated with BMP2-exo/NT, suggesting

522

that the soluble mediators secreted by the differentiating hBMSCs may possibly be

523

the components that magnify the osteogenic signals during tissue repair. Of these, the

524

expression of IL-17A, IL-19, IP-10-/C-X-C motif chemokine 10 [CXCL10], MIP-

525

3α/CCL20, and RANTES/CCL5 significantly increased in the hBMSCs exposed to

526

BMP2-exo/NT than in those treated with Ctr-exo/NT. During bone regeneration, IL-

527

17A expression is upregulated in the repaired tissue after injury, suggestive of its

528

potential role in bone healing. A study using IL-17A-deficient mice demonstrated the

529

impairment in bone regeneration, owing to the decrease in osteoblastic bone

530

formation [69]. The pro-osteogenic effect of IL-17A on MSCs has also been indicated

531

in vitro [70]. IL-19 is a member of the IL-10 subfamily, which plays a critical role in

532

the suppression of inflammation and promotion of angiogenesis [71]. Chemokines are

533

short peptides secreted by several cells [72]. The elevated levels of chemokines IP-10-

534

/CXCL10, MIP-3α/CCL20, and RANTES/CCL5 may also play a pivotal role in the

535

recruitment of specific hematopoietic cells for tissue regeneration [73]. A study

536

indicated the upregulation in the expression of IP-10-/CXCL10 and RANTES/CCL5

537

during the early phase of fracture healing in humans [74]. Although RANTES/CCL5

538

has been shown to act as a chemoattractant for various cell types, its role in bone

539

remodeling has been investigated in a CCL5-deficient mouse model, which showed

540

decreased bone formation and increased osteoclastogenesis [75].This result is

541

collaborated by an in vitro study, wherein the knockdown of the endogenous CCL5

542

expression in hBMSCs significantly impaired osteogenesis [76]. MIP-3α/CCL20 is a

543

member of the MIP family, which is widely expressed in various types of cells. MIP-

544

3α/CCL20 is involved in osteoblast survival and differentiation, as demonstrated by a

545

significant reduction in trabecular bone mass in CCL20-deficient mice [77]. Thus, the

546

increase in the cytokines that we observed in BMP2-exo/NT-stimulated hBMSCs may

547

reflect an interesting but equally important role of BMP2-exo/NT in the regulation of

548

cytokine secretion by hBMSCs during osteogenic differentiation.

549

Conclusion

550

BMP2-stimulated macrophages secret important nano-mediators to regulate

551

intercellular communication. The BMP2/macrophage-derived exosomes may regulate

552

the osteogenic differentiation of MSCs. Our study demonstrates a novel approach of

553

using BMP2-exo to enhance the bio-functionality of titanium nanotubes. Unlike the

554

traditional titanium-based materials, the incorporation of exosomes enables the

555

temporal regulation of MSC differentiation and may create a favorable milieu induced

556

by diverse cytokines for osteogenesis. Given the advanced understanding of the

557

regulatory role of BMP2-exo/NT in osteogenesis, exosome-integrated nanomaterials

558

may serve as novel osteoimmunomodulatory materials. The present study employs an

559

exosome-integrated but single-sized titanium nanotube to understand whether the

560

osteoenvironment generated by exosome-based nanomaterials may influence the

561

differentiation of MSCs. However, further investigations are warranted to understand

562

the influence of titanium nanotubes with various topographic properties. In addition,

563

the present study uses pooled hBMSCs to ensure sufficient number of cells to

564

minimize the inter-donor variability. Further studies using individual donor-derived

565

MSCs are needed to validate the concept for this study.

566 567

Competing interests

568

The authors declare that they have no conflict of interest.

569

570

Author contributions

571

F. W. and YH.Z. carried out the experiment, helped with experimental design and

572

contributed to manuscript preparation. MT.L. contributed to protocol design, guided

573

troubleshooting and edited the manuscript. R.C and Y.X. supervised the project,

574

contributed to experimental design, guided protocol development and edited the

575

manuscript.

576 577

Acknowledgments

578

This work was supported by the National Health and Medical Research Council

579

(NHMRC) Early Career Fellowship (Grant No. 1105035), the National Natural

580

Science Foundation of China (NSFC) Young Scientists Fund (Grant No. 81700969),

581

the National Natural Science Foundation of China (NSFC) (Grant No. 31771025).

582

The authors would like to acknowledge the facilities, and the scientific and technical

583

assistance of Dr Jamie Riches and Rebecca Fieth of the Australian Microscopy &

584

Microanalysis Research Facility at the Central Analytical Research Facility operated

585

by the Institute for Future Environments at the Queensland University of Technology

586

(QUT). Access to CARF is supported by generous funding from the Science and

587

Engineering Faculty, QUT.

588 589

Reference

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Statement of significance

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The clinical application of bone morphogenetic protein 2 (BMP2) is often limited by

819

its side effects. Exosomes are naturally secreted nanosized vesicles derived from cells

820

and play an important role in intercellular communication.

821

The contributions of this study include (1) the demonstration of the potential

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regulatory role of BMP2/macrophage-derived exosomes on the osteogenic

823

differentiation of mesenchymal stromal cells (MSCs); (2) fabrication of titanium

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nanotubes incorporated with exosomes; (3) new insights into the application of

825

titanium nanotube-based materials for the safe use of BMP2.

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