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Tuning Chemistry and Topography of Nanoengineered Surfaces to Manipulate Immune Response for Bone Regeneration Applications Zetao Chen,†,‡,# Akash Bachhuka,§,∥,# Shengwei Han,‡ Fei Wei,‡ Shifeier Lu,‡ Rahul Madathiparambil Visalakshan,∥ Krasimir Vasilev,*,∥ and Yin Xiao*,†,‡ †

Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University and Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, Guangdong, People’s Republic of China ‡ Institute of Health and Biomedical Innovation & the Australia-China Centre for Tissue Engineering and Regenerative Medicine, Queensland University of Technology, Brisbane, Queensland 4059, Australia § ARC Center of Excellence for Nanoscale BioPhotonics, Institute for Photonics and Advanced Sensing, School of Physical Sciences, The University of Adelaide, Adelaide, South Australia 5005, Australia ∥ Future Industries Institute & School of Engineering, University of South Australia, Mawson Lakes, South Australia 5095, Australia S Supporting Information *

ABSTRACT: Osteoimmunomodulation has informed the importance of modulating a favorable osteoimmune environment for successful materials-mediated bone regeneration. Nanotopography is regarded as a valuable strategy for developing advanced bone materials, due to its positive effects on enhancing osteogenic differentiation. In addition to this direct effect on osteoblastic lineage cells, nanotopography also plays a vital role in regulating immune responses, which makes it possible to utilize its immunomodulatory properties to create a favorable osteoimmune environment. Therefore, the aim of this study was to advance the applications of nanotopography with respect to its osteoimmunomodulatory properties, aiming to shed further light on this field. We found that tuning the surface chemistry (amine or acrylic acid) and scale of the nanotopography (16, 38, and 68 nm) significantly modulated the osteoimmune environment, including changes in the expression of inflammatory cytokines, osteoclastic activities, and osteogenic, angiogenic, and fibrogenic factors. The generated osteoimmune environment significantly affected the osteogenic differentiation of bone marrow stromal cells, with carboxyl acid-tailored 68 nm surface nanotopography offering the most promising outcome. This study demonstrated that the osteoimmunomodulation could be manipulated via tuning the chemistry and nanotopography, which implied a valuable strategy to apply a “nanoengineered surface” for the development of advanced bone biomaterials with favorable osteoimmunomodulatory properties. KEYWORDS: osteoimmunomodulation, surface chemistry, surface nanotopography, inflammation, bone regeneration clastogenesis, and mediating osteogenesis.2−4 Bone biomaterials with favorable osteoimmunomodulatory properties should modulate an adequate immune response, releasing osteogenic and osteoclastogenic factors that may induce a beneficial balance of osteogenesis over osteoclastogenesis for functional bone regeneration.5,6 It is of great significance to manipulate

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steoimmunomodulation, a concept arising from the convergence of osteoimmunology and immunomodulation, is proposed to be a vital biological property of bone biomaterials for mediating osteogenic outcomes.1 The basic principles underlying this concept are the immunomodulatory capacity of bone biomaterials and the important roles of generated osteoimmune environments on bone dynamics. Osteoimmunomodulation provides a valuable strategy for developing advanced bone biomaterials with multifactorial effects on manipulating immune response, regulating osteo© 2017 American Chemical Society

Received: November 20, 2016 Accepted: April 17, 2017 Published: April 17, 2017 4494

DOI: 10.1021/acsnano.6b07808 ACS Nano 2017, 11, 4494−4506

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Figure 1. AFM data analysis. (A, B) 2D and 3D images of uncoated GCS and GCS modified with 16, 38, or 68 nm nanoparticles, respectively. (C, D) RMS and number of particles per μm2 on different surfaces. (E−G) XPS results: (E) gold atomic concentration; (F) nitrogen/carbon ratio; (G) oxygen/carbon ratio of different chemically and nanotopographically modified surfaces.

tion and complement activation can be attenuated by nanotopography.16−19 These multifactorial effects of nanotopography make it an excellent target for modifying the surface properties of bone materials. Therefore, it would be of great interest to utilize nanotopography to modify bone materials’ surface properties, thereby manipulating osteoimmunomodulation to enhance bone regeneration. This approach will lead to a valuable and more advanced nanotopography-based strategy for bone biomaterial development. To achieve this aim, the osteoimmunomodulatory effect of nanotopography must be understood. Although the effects of nanotopography on bone cells or immune cells have been investigated, these studies focused on individual interactions, while neglecting the effects of altered immune environment on bone cell responses, which is an essential factor of osteoimmunomodulation. To bridge this knowledge gap, we developed surfaces with controlled nanotopography and tailored surface chemistry. The interactions of these surfaces with immune cells (macrophages) were investigated, and the osteogenic effects of the synthetic osteoimmune environment on bone marrow stromal cells (BMSCs) were assessed. These studies were performed to demonstrate the osteoimmunomodulatory effects of nanotopography and understand the interplay of surface chemistry and nanotopography that regulates osteoimmunomodulation.

osteoimmunomodulation to obtain favorable repair outcomes. Due to their exogenous nature, bone biomaterials are recognized by the host’s immune system as a foreign body, eliciting significant effects on immune cells and the subsequent immune response.7,8 The generated immune environment varies with the differing properties of biomaterials, including their surface properties, particle sizes, porosities, released bioactive ions, etc. Therefore, osteoimmunomodulation may be manipulated via regulating the properties of the biomaterials. The interactions between cells and surfaces are crucial for cellular behaviors and functions. By applying controlled surface topography, processes including the kinetics and force of cellular adhesion to a surface, migration, proliferation, and differentiation of cells, and deposition of extracellular matrix proteins and minerals might all be obtained.9−11 On the basis of the importance of surface nanotopography, nanofabrication techniques were introduced to modify the surface properties of bone biomaterials and enhance functional outcomes. A number of studies have investigated the behaviors of bone cells, especially osteoblastic lineage cells, on differing nanotopographies.9,11−14 The adhesion, migration, proliferation, and differentiation of osteoblastic cells were successfully regulated by modifying nanotopography.9,12−14 The mechanisms by which nanotopography mediates osteogenesis may involve the coactivation of adhesion and bone morphogenic protein signaling pathways in mesenchymal stromal cells.11 In addition to its effects on bone cells, nanotopography also has a significant effect on immune cells. Nanotopographies can control the morphology, adhesion, proliferation, and inflammatory response of macrophages.15,16 Additionally, inflamma-

RESULTS AND DISCUSSION Tuning Chemistry and Topographies of Nanoengineered Surfaces. To fabricate nanoengineered surfaces with 4495


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Figure 2. Growth and morphology of macrophages on different nanotopographies were observed by light microscopy (A, D). Macrophages grown on the AApp/ACpp surfaces without nanotopographies showed an elongated spindle shape. Cell shapes in the 16AApp group were similar to shapes in the AApp group, while macrophages in the 38AApp and 68AApp groups were more rounded with pseudopodia. Cells grown on all three ACpp-coated surfaces with nanotopography were rounded and had “star”-like shapes with apparent pseudopodia. The contact of macrophages with nanotopographies was demonstrated by SEM and TEM in the top view and side view, respectively (SEM: B, E; TEM: C, F). The extended pseudopodia appeared to attach well to AApp, 16AApp, and all ACpp tailored surfaces showing a large cell attachment area, while on the 38AApp and 68AApp surfaces, the extended pseudopodia had a much smaller contact area (B, E). The interfaces between cell membranes and nanorough surfaces were visualized by TEM (C, F). The gold nanoparticles stayed intact and attached to the surface. Cell membranes integrated well with the polymer films, which did not fold and fit into the interparticle spaces, but lined the top of the particles.

controlled nanotopography and chemistry, a combination of plasma polymerization and electrostatic self-assembly techniques was employed. To generate controlled surface nanotopographies, substrata were first modified with a thin plasma polymer film (∼20 nm) deposited from an allylamine (AApp) vapor, thus generating coatings rich in amine groups. AApp coatings tend to carry a positive charge in aqueous medium at pH < 8.20,21 AApp-modified surfaces were immersed in a solution of gold nanoparticles (AuNPs) of nearly monodispersed sizes of 16, 38, or 68 nm for a predetermined time. These nanoparticles were premodified with a monolayer of mercaptosuccinic acid (MSA), which provides a negative charge and thus facilitates a strong electrostatic attachment of the nanoparticles to AApp-coated surfaces, which carry a positive charge in aqueous medium.20 The immobilization of gold nanoparticles in such a fashion led to the generation of surface nanotopographies of controlled magnitudes. However, similar to substrates used in other biological studies, the model surfaces prepared in this manner possessed mixed chemistry consisting of carboxyl acid groups on the surface of the nanoparticles and nitrogen/amine chemistry on the surface of the AApp. To address this issue, a 5 nm thin plasma polymer coating was added onto the nanotopographically modified surfaces. From previous detailed studies, plasma polymer films thicker than 5 nm are known to be continuous and pinhole-free and provide a uniform outermost surface chemistry.22,23 AApp and ACpp (acrylic acid) films were used to coat the nanotopographically modified surfaces. These polymeric films were chosen because they resemble the common chemical functionalities present in biological tissues. Smooth AApp and ACpp coatings were used as controls. The water contact angle of the smooth controls was 68.5 ± 3.5° and 50 ± 2.6° for AApp and ACpp coatings, respectively. The nanotopographically and chemically modified surfaces elicited significant effects on wettability, with the water contact

angle increasing as expected from Wenzel and Cassie theories and accurately predicted by the recently proposed Vasilev− Ramiasa equation.24 The 16 nm gold nanoparticles appeared to have the most significant effect on increasing the water contact angles of both AApp and ACpp surfaces (16AApp: ∼80 ± 3.1°; 16 ACpp: ∼62.5 ± 3.2°), followed by the 38 and 68 nm nanoparticles. This effect was due to the greater number of surface-bound 16 nm diameter nanoparticles (Figure 1A, B, and D). Atomic force microscopy (AFM) was used to characterize the model surfaces with controlled nanotopography and tailored surface chemistry. The AFM images in Figure 1A and B demonstrate that the nanotopography was well preserved after the addition of the 5 nm thin plasma polymer layer. The RMS (root mean square) was increased most significantly in the 68 nm topographies (RMS: ∼15 nm), followed by the 38 and 16 nm surfaces (RMS: ∼10 and ∼5 nm), as demonstrated in Figure 1C and D, showing the distribution of particles across the surface. The particle immobilization densities decreased with the increase in particle size (16AApp/ACpp > 38AApp/ ACpp > 68AApp/ACpp). X-ray photoelectron spectroscopy (XPS) was employed to determine the elemental composition of the chemically and nanotopographically modified surfaces. Figure 1E displays the successful surface modification with gold nanoparticles and tailored chemistries of the model substrates. This figure shows the gold atomic concentration after immobilization of different sized nanoparticles on the AApp surfaces before and after addition of an overcoating. The detection of gold (although in reduced amounts) after coating with AApp or ACpp further confirmed that the polymer overcoat was thinner than the sampling depth of XPS, which is approximately 10 nm. Nitrogen/carbon (N/C) ratios determined from the XPS spectra of the samples used in this study were also investigated (Figure 1F). As expected, the nitrogen surface concentration was greater for samples where the outermost chemistry was 4496


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Figure 3. Inflammatory response of macrophages stimulated by different surface chemistries and nanotopographies. (A) Effects of the surface chemistry on the expression of inflammatory genes (IL-1ra, TNFα, IL-18, IL-1β, and IL-6) detected by RT-qPCR. *Significant difference (P < 0.05) compared to the LPS group. (B) Effects of nanotopographies on the expression of inflammatory genes detected by RT-qPCR. *Significant difference (P < 0.05) compared to the AApp/ACpp group. (C) Effects of the surface chemistry on the expression of macrophage marker genes (M1 markers: CD11c and CD86; M2 markers: CD206 and arginase) detected by RT-qPCR. *Significant difference (P < 0.05) compared to the LPS group. (D) Effects of nanotopographies on the expression of macrophage marker genes detected by RT-qPCR. *Significant difference (P < 0.05) compared to the AApp or ACpp group. (E) Protein expression of IκB, iNOS, and CD86 by Western blotting. IκB is an inhibitor of NFκB, the high expression of which indicates an anti-inflammatory response. *Significant difference (P < 0.05) compared to LPS group.

controlled addition of AApp/ACpp and gold nanoparticles tuned the surface composition, charge, wettability, roughness, and topographies. In the following, we investigate how these properties influence the biological behaviors of immune cells (macrophages). Cell Shapes of Macrophages on Different Nanosurfaces. On all tested surfaces, macrophages attached, spread, and grew well (Figure 2, Supplementary Figures 1 and 3). However, macrophages possessed different cell morphologies on different surfaces (Figure 2A,D, Supplementary Figure 2). Macrophages grown on the AApp/ACpp surfaces without nanotopographies showed a more elongated spindle shape. The addition of nanotopography effectively changed the cell shapes. Cells grown on all three ACpp-coated surfaces containing nanotopography were more rounded with apparent pseudopodia. Interestingly, cell shapes in the 16AApp group were not significantly different than shapes in the AApp group without nanotopography, while in the 38AApp and 68AApp groups, macrophages tended to be rounded with more pseudopodia. These morphologic changes in the 38/68AApp groups were not as evident as those in the 16/38/68ACpp groups. Adhesive cues play a vital role in regulating cell shapes. Nanotopographies have a contact guidance effect, which is a

based on AApp. The detection of nitrogen on ACpp-coated nanotopographically modified samples was due to the underlying AApp coating, which was used to bind the gold nanoparticles. Pure ACpp coating did not contain nitrogen, as visible in Figure 1F and G, showing the oxygen/carbon (O/ C) ratio measured by XPS. Pure ACpp- and ACpp-coated samples containing nanotopography had the highest O/C ratio, which was expected based on the precursor chemistry and consistent with published studies.10,24,25 Overall, these data confirm the successful surface modification of the model substrata used in this study, in terms of chemistry and nanotopography. Effects of Modified Surface Chemistry and Nanotopographies on Manipulating the Immune Response of Macrophages. The interface between nanotopographies and biological systems comprises dynamic physicochemical interactions, kinetics, and thermodynamic exchanges between the nanomaterial surface and biological components (e.g., proteins, body fluid, intracellular organelles, and cell membranes).26 Via these interactions, the physicochemical and mechanical signals from the surface nanotopography are translated into biological signals, thereby modulating the local biological microenvironment and cellular responses. The 4497


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Figure 4. Activation of autophagy in macrophages grown on different nanotopographies. Autophagy is an important intracellular process, which participates in the modulation of inflammation, especially in downregulating IL-18 and IL-1β. (A) Effects of the surface chemistry on the expression of autophagy pathway related genes (Atg5, Atg7, P62, LC3A, and LC3B), detected by RT-qPCR. *Significant difference (P < 0.05) compared to the LPS group. (B) Effects of nanotopographies on the expression of autophagy pathway related genes. *Significant difference (P < 0.05) compared to the AApp or ACpp group. (C) Expression of autophagy pathway related proteins (Atg3, Atg7, P62, LC3, and Beclin-1) detected by Western blotting with quantitative analysis done by ImageJ. *Significant difference (P < 0.05) compared to the LPS group. (D) Intracellular autophagic structures were observed in macrophages from the 16AApp group by TEM. Damage of mitochondria (white arrow) was observed next to a gold nanoparticle (GN), which can initiate autophagy. The elongation of a phagophore (red arrow) was activated to phagocytize damaged mitochondria, then forming an autophagosome. The autophagosome further fused with a lysosome to generate an autophagolysosome that could clean up the damaged mitochondria (yellow arrow) to maintain the intracellular homeostasis.

Attachment of Macrophages to Different Nanosurfaces. The scanning electron microscope (SEM) images display the different affinities of macrophages to different surfaces (Figure 2B,E, Supplementary Figure 3). Macrophages seemed to attach and extend well on the AApp, 16AApp, and all ACpp tailored surfaces, having large cell attachment areas, while on the 38AApp and 68AApp, the extended pseudopodia of macrophages had much smaller contact areas. The interfaces between cell membrane and nanorough surfaces were visualized by transmission electron microscopy (TEM) (Figure 2C,F). The gold nanoparticles stayed intact and attached to the surface after macrophage-mediated degradation. Cell membranes integrated well with the polymer films. The membranes did

phenomenon in which cells elongate and preferentially follow along the architectures of the substrates.27−29 In the present study, we demonstrated that surface chemistry itself (AApp vs ACpp) had limited effects on cell shape. However, when the topographies were added, the cell shapes changed, which could be due to the contact guidance effect from the topographies. Although surface chemistry itself does not elicit a significant effect, it is reported to play regulatory roles on the contact guidance effect.27,28 AApp and ACpp chemistries also generated a positive regulatory effect on the cell morphologic changes mediated by nanotopographies, as implied from the different cell shapes on surfaces with the same nanotopographies but different chemistries. 4498


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ACS Nano not fold and fit into the interparticle spaces, but lined the top of the particles. A number of studies have proved that surface wettability and charge affect cell adhesion, shape, and spreading, via affecting adhesion protein (such as fibronectin, laminin, and collagen fibers) absorption and structure.28,30,31 It is proposed that cell affinity is enhanced on hydrophilic surfaces, which is consistent with our findings. Compared with the four AApp groups, all four ACpp-coated surfaces, which were more hydrophilic, showed better cell affinities.32,33 In addition to the wettability, it is reported that roughness affects the attachment of immune cells, with cell affinities increasing with the rise of surface roughness.34 The greater surface roughness of the 68AApp surfaces could be the mechanism that mediates superior cell spreading compared with the 38AApp group. Inflammatory Response of Macrophages. In addition to the effects on cell attachment, spreading, and shape, nanoscale variations and surface signals could also be transduced into the cytoplasm, resulting in significant biological responses. Adhesive cue-mediated cell shape change is an effective indicator of the inflammatory response of macrophages.35 The shape changes of macrophages under the stimulation of nanosurfaces with different chemistries and topographies strongly imply a possible modulation of the immune environment. Therefore, we further investigated the immune environment generated by macrophages on nanoengineered surfaces with controlled chemistries and topographies. Effects of Surface Chemistry on Modulating Inflammation. Surface chemistry controls the profile and structure of absorbed protein. Amine groups (−NH2) favor the absorption of fibrogen, fibronectin, and albumin; however, these proteins are more easily eluted from surfaces with a carboxyl group (−COOH).21,36,37 The binding of these proteins might expose inflammatory protein sequence fragments, facilitating the attachment of immune cells, the fusion of foreign body giant cells (FBGCs), and the subsequent formation of fibrosis.1 This indicates the AApp surface chemistry should be more proinflammatory than the ACpp chemistry. Interestingly, when liposaccharide (LPS) was applied to activate macrophages, the anti-inflammatory effect of AApp on LPS-mediated inflammation was stronger than the effect of ACpp (Figure 3A,C). Genes encoding pro-inflammatory cytokine were downregulated by the AApp surface more dramatically than the ACpp surface (Figure 3A). We also investigated the expression of pro-inflammatory M1 and pro-healing M2 phenotype markers [M1: CD11c, CD86, and iNOS (nitric oxide synthases); M2: CD206 and arginase], to further define the function of macrophages on different surface chemistries. The expression of M1 markers was inhibited, whereas expression of M2 markers was enhanced on AApp (Figure 3C,E). On ACpp the inhibitory effect on the expression of M1 markers was less notable, and no significant increase was observed in the expression of M2 markers. These data imply that the AApp chemistry switched the macrophages toward a more prohealing extreme. Collectively, these results indicate that in addition to direct modification of protein absorption and structure other mechanisms underlie the surface chemistry-mediated immunomodulation. We hypothesized that changes in the intracellular microenvironment and the subsequent signaling cascade might be important. Autophagy plays a vital role in maintaining intracellular homeostasis and actively participates in the immune response. The expression of nitric oxide (Figure 3E),

an inhibitor that blocks autophagy via inactivating Beclin-2 phosphorylation and dissociation from Beclin-1,38,39 was downregulated in both AApp and ACpp. Therefore, we hypothesized that the immunomodulatory effects of surface chemistry could be associated with the activation of autophagy. Compared to ACpp, AApp had a more significant effect on upregulating the expression of autophagy pathway components [LC3A/B (microtubule-associated protein 1 light chain 3), Beclin-1, Atg3 (autophagy-related gene 3), Atg5, Atg7, and P62 (also known as SQSTM1, sequestosome 1)], indicating improved activation of autophagy on AApp (Figure 4A,C). LC3 fluorescent staining further confirmed the activation of autophagy on AApp, showing more LC3 (+) cells in comparison with that on ACpp (Supplementary Figure 4). This activation of autophagy might have a significant inhibitory effect on inflammation by reducing the activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells).40−42 Inflammasomes are cytoplasmic complexes responsible for the processing and secretion of IL-1β and IL-18.43,44 Autophagy suppresses inflammasome activation,45,46 which may be responsible for the observed inhibition of IL-1β and IL-18 expression (Figure 3A). Synergetic Effects of Surface Nanotopography on Modulating Inflammation. Together with surface chemistry, nanoscale surface topography seemed to have synergetic modulatory effects on the inflammatory response. The addition of nanotopographies of 16, 38, or 68 nm further inhibited the inflammatory response of macrophages, especially affecting gene expression of the pro-inflammatory cytokines IL-18, IL1β, and IL-6 (Figure 3B). An M2 marker (arginase) was also upregulated in response to the added nanotopography (Figure 3D), while an M1 marker (iNOS) was downregulated (Figure 3E). It was also found that the expression of IκB (inhibitor of nuclear factor of kappa-light-chain-enhancer of activated B cells) was upregulated (Figure 3E). IκB is an inhibitor of the nuclear factor NF-κB, which plays an important role in the expression of pro-inflammatory genes.47 The upregulation of IκB may inhibit the activity of NF-κB, thereby impeding inflammation. As previously discussed, the observed synergetic modulatory effect of surface nanotopography could also be related to the regulation of autophagy, the activation of which may inhibit inflammation. The expression of autophagy pathway components (LC3A/B, Beclin-1, Atg3, Atg5, Atg7, and P62) was mostly enhanced by nanotopography more than by surface chemistry alone (Figure 4B,C). Particularly for ACpp, the synergetic effect of nanotopography on activating autophagy was highly significant. The autophagic structures in macrophages grown on different nanoengineered surfaces were visualized by TEM (Figure 4D, Supplementary Figure 5). Damage of intracellular organelle mitochondria next to gold nanoparticles was observed, which activated the initiation and elongation of phagophores and formed autophagosomes. Autophagosomes further fused with lysosomes to generate autophagolysosomes that could clean up the damaged mitochondria to maintain intracellular homeostasis (Figure 4D, Supplementary Figure 5). This suggested that the activation of autophagy by different nanotopography may be due to the damage of organelles, such as mitochondria. These possible intracellular mechanisms in macrophages upon the stimulation of nanoengineered surfaces with different surface chemistry and topographies are illustrated and summarized in Supplementary Figure 9. 4499


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Figure 5. (A) Effects of the surface chemistry on the expression of osteoclastic activity related genes detected by RT-qPCR. *Significant difference (P < 0.05) compared to the LPS group. (B) Effects of nanotopographies on the expression of osteoclastic activity genes detected by RT-qPCR. *Significant difference (P < 0.05) compared to the AApp or ACpp group. (C) Effects of the surface chemistry on the expression of osteogenic factors (BMP2, BMP6, WNT10b, and OSM), an angiogenic factor (VEGF), and a fibrogenic factor (TFGβ1), detected by RTqPCR. *Significant difference (P < 0.05) compared to the LPS group. (D) Effects of the nanotopographies on the expression of osteogenic, angiogenic, and fibrogenic factors, detected by RT-qPCR. *Significant difference (P < 0.05) compared to the AApp or ACpp group.

surface chemistry regulates osteoclastic activities. The addition of nanotopographies changed the gene profiles. Synergetic effects were observed on ACpp, with all osteoclastic activity genes being upregulated and MMP9 being dominantly expressed after adding nanotopographies of 16, 38, or 68 nm gold nanoparticles (Figure 5B). Different results were observed on AApp. The addition of 16 nm nanotopography favored the expression of tartrate-resistant acid phosphatase (TRAP) and CR, whereas MMP9 was the most significantly upregulated gene on 38 and 68 nm nanotopographies (Figure 5B). Both surface chemistry and nanotopography showed significant effects on regulating osteoclastic activities, which may affect the degradation of materials and remodeling of newly formed bone. In this context, tuning surface chemistry and nanotopography may be a promising strategy to manipulate osteoclastic activities. Osteogenic, Fibrogenic, and Angiogenic Factor Expression in Macrophages. Macrophages can release osteogenic, fibrogenic, and angiogenic factors to actively participate in osteogenesis, fibrosis, and angiogenesis.1 To fully define the osteoimmune environment, these factors should be assessed.1 The AApp surface appeared to be more favorable for the expression of osteogenic factors (bone morphogenetic protein 2, BMP2), whereas ACpp stimulated greater expression of angiogenic factors (VEGF) and fibrogenic factors (TGF-β1) (Figure 5C). These data suggest that AApp could be more osteogenic and ACpp more fibrogenic. ACpp appeared to act synergistically with nanotopographies to enhance the expression of angiogenic factors (VEGF) and fibrogenic factors (TGF-β1) (Figure 5D). All osteogenic factors [BMP2/6, wingless-type MMTV integration site family member 10B (Wnt10b), and Oncostatin M (OSM)] were upregulated on the AApp surface after adding 16 nm nanotopography, indicating that the 16AApp group may be the best of the eight groups at stimulating osteogenesis (Figure 5D). The gene expression profile of the 38AApp group was similar to that of the ACpp groups, with greater expression of angiogenic factors (VEGF) and fibrogenic factors (TGF-β1) (Figure 5D). The

Modulation of the biomaterial-mediated inflammatory response is of great importance. The immune response not only determines biocompatibility but also participates in regulating the activities of tissue-resident cells, thereby affecting tissue restoration outcomes. Excessive inflammation may lead to the formation of a fibrous capsule, which prevents the bone cells from contacting and integrating with the implants, resulting in the failure of bone regeneration, whereas a proper inflammatory response may enhance the recruitment and differentiation of osteoblastic lineage cells, thus improving osteogenesis.5,6 Surface chemistry and nanotopographies could be of great value for immunomodulating bone tissue regeneration. Osteoclastic Activities of Macrophages. Successful bone regeneration requires effective osteoclastic activities.3 Improper osteoclastic activities may lead to fast degradation and affect the long-term performance of implant components. In addition, the low quality of the newly formed bone tissue (poor bone mass and density) may result in unsatisfactory mechanical performances. Therefore, it is very important to trigger proper osteoclastic activities during bone regeneration. Pro- and antiinflammatory cytokines play important roles in regulating the expression of osteoclastic factors [macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL)], thus affecting osteoclastogenesis.48 The osteoclastogenic differentiation and the osteoclastic activities of preosteoclast macrophages could be regulated under different nanoengineered surface-mediated immune environments. It was found that macrophages grown on different nanoengineered surfaces could merge into multinucleated cells with 3−5 nuclei (Supplementary Figure 6), suggesting the formation of osteoclastic-like cells.49 The osteoclastic activities of macrophages on different surface chemistries and nanotopographies were then assessed. AApp and ACpp induced different patterns of osteoclastic genes. AApp favored the expression of calcitonin receptor (CR), while expression of matrix metalloproteinase-9 (MMP9) was dominant in the ACpp group (Figure 5A). This implies that 4500


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Figure 6. In vitro osteogenic differentiation of BMSCs. (A) Alizarin red staining was used to demonstrate the mineralized nodules formed by BMSCs’ exposure to media conditioned by macrophages cultured on different nanotopographic surfaces. (B) Quantitative analysis of alizarin red results. *Significant difference (P < 0.05) compared to AApp group. (C) Expression of osteoclastogenesis factors, detected by RT-qPCR. RANKL is an osteoclastogenic factor, while OPG is an inhibitor. *Significant difference (P < 0.05) compared to the LPS group.

expression profile of the 68AApp group featured higher expression of OSM (Figure 5D). The balance of fibrosis and osteogenesis plays a vital role in determining the fate of bone biomaterials, either forming new bone or generating a fibrotic capsule. Generating an osteogenic microenvironment is of great importance for bone regeneration. By controlling surface chemistry and nanotopography, the osteoimmune environment (including inflammatory cytokines, osteoclastic activities, osteogenic, fibrogenic, and angiogenic factors) was significantly modified. This implies that tuning the surface chemistry and nanotopography could be a promising strategy to manipulate osteoimmunomodulation, thereby enhancing osteogenesis. The mechanisms underlying the immune reactions stimulated by surface chemistry and nanotopography are still largely unknown. In this study, the surface chemistry and nanotopography significantly affected cell shape, which is associated with the inflammatory response of macrophages. Autophagy activation was also observed as an intracellular reaction to maintain homeostasis. These data indicate that the modulation of cell shape and activation of autophagy may be key factors in transforming different mechanical and physicochemical signals into different biological signals, which then initiate immune cell responses (Supplementary Figure 9). The results of this study may bridge the gap between the immune response and nanotopography, thus stimulating systemic investigation on these poorly understood mechanisms. Osteogenic Differentiation of BMSCs in Different Osteoimmune Environments. After defining the osteoimmune environment generated by the interaction of macrophages and nanoengineered surfaces, the next step was to test the effect of these microenvironments in osteogenesis. Therefore, we then investigated the osteogenic differentiation of BMSCs in different osteoimmune environments. BMSCs grew well in these environments (Supplementary Figure 7). The cells cultured on 38AApp, 68AApp, 38ACpp, and 68ACpp clumped

together to form mineralized nodules (Supplementary Figure 7). Alizarin red staining demonstrated that there was no significant difference in the formation of mineralized nodules between the AApp and ACpp groups, indicating that these substrates had similar effects on regulating the osteogenic differentiation of BMSCs (Figure 6A,B). With the addition of nanotopography, osteogenic differentiation was significantly enhanced, with a size-dependent effect. The surface chemistry elicited a significant effect on nanotopography-mediated osteogenesis. ACpp was more favorable than AApp for nanotopography-mediated osteogenesis. Of all eight groups, the 68ACpp group was the best at forming mineralization nodules (Figure 6A,B). Osteogenic factors, including BMP2/6, Wnt10b, and OSM, can activate three osteogenic pathways, i.e., the BMP, Wnt, and OSM pathways, to synergistically enhance osteogenesis. Therefore, an osteoimmune environment that includes upregulation of these factors would be beneficial for osteogenesis. Of all eight groups, the 16AApp group was the most effective at improving the expression of these factors (Figure 5D), suggesting that 16AApp should be the prime surface for osteogenesis. Interestingly, a 16AApp and macrophage osteoimmune environment did not enhance osteoblastic differentiation (Figure 6A, Supplementary Figure 7), although conditioned medium from these cultures, containing secreted BMP2/6, did stimulate upregulation of the BMP pathway in BMSCs (Supplementary Figure 8). This inconsistency between the expression of osteogenic factors and the osteogenic differentiation of BMSCs implies that other more important factors were involved. Inflammation has multiple effects on bone dynamics, and its regulatory effects are complicated.50,51 It can be beneficial or detrimental for osteogenesis. Excessive inflammation often has destructive effects on bone, whereas inflammatory processes also trigger new bone formation during the initial phase of bone fracture repair, which can be impaired by the application of 4501


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Scheme 1. Introduction of allylamine (AA) or acrylic acid (AC) and gold nanoparticles (16, 38, 68 nm) significantly modified surface properties, including the chemical composition, surface charge, topographies, wettability, and roughness. These physicochemical signals led to differing cell affinities, shapes, and activated intracellular autophagy. These cell changes resulted in functional responses, including the release of inflammatory cytokines and osteogenic, angiogenic, and fibrogenic factors and the regulation of osteoclastic activities. The generated osteoimmune environment subsequently affected the osteogenic differentiation of BMSCs and the formation of new bone.

pharmacological anti-inflammatory agents.52−54 The expression profile of inflammatory cytokines should be taken into consideration when assessing osteogenesis. After the addition of nanotopography, the expression profile of inflammatory cytokines was significantly changed, with a dramatic downregulation of IL-1β and IL-18 (Figure 3B). Interestingly, 16AApp had a less significant inhibitory effect than the other nanotopographically modified surfaces (38/68 AApp and 16/38/68 ACpp). Correlating these differing expression profiles of inflammatory cytokines (Figure 3) with the different osteogenic differentiation outcomes (Figure 6) indicates that downregulation of IL-1β and IL-18 could be an important mechanism underlying the nanotopographic enhancement of osteogenesis. Higher expression of IL-1β and IL18 in the 16AApp group might attenuate the ability of the osteogenic factors to enhance osteogenic differentiation, which explains the inconsistency between the expression of osteogenic factors and the osteogenic differentiation of BMSCs in the 16AApp group. This is in line with a prior study in which IL-1β had a dose-dependent inhibitory effect on the osteogenic differentiation of MSCs, with higher doses having a higher inhibitory effect.55 We also compared the synergetic effects of surface chemistry on nanotopography-mediated osteogenesis and found that all ACpp groups were superior to the AApp groups at improving osteogenic differentiation. The major difference in the expression profiles of inflammatory cytokines between 16/38/ 68AApp and 16/38/68ACpp was the expression of IL-6. 16/

38/68AApp appeared to be more effective than 16/38/68ACpp at inhibiting expression of IL-6 (Figure 3B). As a proinflammatory cytokine, IL-6 is typically considered to be an osteoclastogenic factor, shifting the balance of osteogenesis and osteoclastogenesis toward the latter, thus enhancing bone degradation. However, a beneficial effect of IL-6 on osteogenesis has also been documented.56,57 The differentiation of osteoblastic lineage cells is enhanced by IL-6 in vitro.56 IL-6 may also improve the osteogenic differentiation of adiposederived stromal cells,57 indicating the important roles of IL-6 in osteogenesis. Therefore, 16/38/68ACpp may enhance osteogenesis by promoting the proper concentration of IL-6, in comparison with 16/38/68AApp. It should be noted that the effects of inflammatory cytokines on osteogenesis are very complicated, with the response being related to the dose and combinatorial effects of different cytokines. To thoroughly understand the mechanisms that underlie substrate-mediated osteogenesis, the complete inflammatory profiles of activated macrophages on nanoengineered substrates should first be identified, to pinpoint the relevant cytokines. Then, gain- and loss-of-function studies should be carried out to verify the roles of the most important cytokines. The osteoimmune environment modulated by controlled surface chemistry and nanotopographies significantly affects the osteogenic differentiation of BMSCs, proving that tuning surface chemistry and nanotopography is a rational approach for manipulating osteoimmunomodulation. By tuning surface chemistry and nanotopography, immune responses may be 4502


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ment on bone dynamics, bone cells’ response can be regulated and directed into desired extremes by differently modulated immune environments (Scheme1). Therefore, manipulating osteogenesis by modifying the chemical and topographical properties of the nanosurface and modulating the corresponding immune reactions may be a valuable tool for orthopedic applications.

guided into therapeutic ones that favor osteogenesis, thereby enhancing functional bone regeneration. Osteoclastogenic Factors Released from BMSCs. In bone, osteoblastic cells are a source of osteoclastogenesis-regulating factors [osteoprotegerin (OPG) and RANKL].58 RANKL/ RANK/OPG is a key pathway in regulating the differentiation and maturation of osteoclasts, with RANKL as an activator and OPG as an inhibitor. Therefore, we investigated the expression of RANKL and OPG from stimulated BMSCs, to assay the possible effects of the engineered surfaces on osteoclastogenesis. RT-qPCR results indicated that the AApp, ACpp, 38ACpp, and 68ACpp groups exhibited no significant changes in the balance of RANKL and OPG expression (Figure 6C). However, OPG expression was favored on 16AApp, but RANKL was more dominant on 68AApp and 16ACpp. This suggests that osteoclastogenesis can be regulated by modifying surface chemistry and topographies. Valuable Strategy to Manipulate Osteogenesis via Tuning Surface Chemistry and Nanotopographies. Precise tuning of surface chemistry and nanotopographies provides an opportunity to generate desired biophysicochemical and mechanical signals to modulate physiological responses. In this paper, we demonstrated a simple method for tailoring these surface properties. Surface-immobilized nanoparticles can create nanotopographies with controlled height and lateral spacing.24,59 Many techniques for fabricating nanotopography result in undefined (even unknown) surface chemistry, but our technology is effective in tuning the chemical function of the outermost surface. This was achieved by placing a thin plasma polymer film of desired, known, and defined surface properties and thickness on top of the nanoparticle-modified substrata.24,59 This film allowed the retention of the targeted scale of nanotopography.24,59 To successfully translate contact-mediated cues into biological signals and modulate the immune response, immune cells must have a high “sensitivity” to such mechanical cues. The plasticity of immune cells determines how “sensitive” the immune system is in responding and adjusting to the nanotopography-mediated local environment change. Therefore, the immune system should be modulatory and can be manipulated by modifying the properties of biomaterials. Conversely, the immune system is a highly flexible network that serves as a guardian of tissue integrity and is adapted to the nature of the local microenvironment.60 In particular, macrophages have been the most highly studied immune component due to their high diversity and plasticity. In response to various stimulations, macrophages can convert to different phenotypes, including the M1 phenotype, which is characterized by high expression of pro-inflammatory mediators and production of reactive nitrogen and oxygen intermediates. Alternatively, the activated M2 profile participates in encapsulation of parasites and promotes angiogenesis and matrix remodeling.61−63 M2 phenotypes can also be divided into three different subtypes based on cytokine secretory profiles and surface receptor expression: M2a (anti-inflammatory), M2b (immune-regulatory), and M2c (remodeling). The high plasticity of immune cells is another vital factor that contributes to the successful carrying out of this proposed strategy. The highly controllable and regulatory properties of nanosurface chemistries and topographies and the plasticity of immune cells provide a solid basis for developing immunomodulation strategies via tuning nanosurface chemistries and topographies. Due to the importance of the immune environ-

CONCLUSIONS Nanoengineered surfaces with tailored chemistry (amine rich or acid rich) and topography (height of 16, 38, or 68 nm) modulated immune cell response and generated different osteoimmune environments that influence osteogenesis. The surface chemistry and nanotopography applied in this study synergistically inhibited inflammation, switching macrophages toward the prohealing extreme. The osteoclastic activities and expression of osteogenic, angiogenic, and fibrogenic factors were also regulated, which subsequently affected the osteogenic differentiation of BMSCs. The immune environment generated by 68ACpp may be the prime environment for improving osteogenesis. Osteoimmunomodulation may be manipulated via tuning surface chemistry and nanotopography for the development of advanced bone biomaterials that could direct inflammation, osteogenesis, and osteoclastogenesis. METHODS Preparation of Model Substrata with Controlled Surface Nanotopography and Tailored Surface Chemistry. Materials. Allylamine (AA) (98%, Aldrich), acrylic acid (AC) (99%, Aldrich), hydrogen tetrachloroaurate (99.9985%, ProSciTech), trisodium citrate (99%, BHD Chemicals, Australia Pty. Ltd.), and 2-mercaptosuccinic acid (97%, Aldrich) were used as received. Plasma Polymerization. Surfaces were cleaned and modified with different chemistry using a custom-built plasma reactor with a 13.56 MHz plasma generator.64 A high power of 50 W was applied for 2 min under an oxygen atmosphere to clean all substrates prior to coatings. For chemical modification, allylamine and acrylic acid were employed. Powers of 40 and 10 W were used at a pressure of 0.2 mbar for 2 min to obtain plasma-polymerized allylamine (AApp) and acrylic acid (ACpp) surfaces of thicknesses 23 and 20 nm, respectively. To coat the surfaces with 5 nm coatings of AApp and ACpp, the deposition time was reduced to 20 s. Synthesis of Gold Nanoparticles. Hydrogen tetrachloroaurate (HAuCl4) was employed to synthesize gold nanoparticles. The amount of 1% trisodium citrate was varied from 1 to 0.3 mL to create particles of 16, 38, and 68 nm diameter, respectively.65 Surface modification of these nanoparticles was performed using 2mercaptosuccinic acid.66 Immobilization of AuNPs. Gold nanoparticles of different size (16, 38, and 68 nm) were immobilized on plasma polymerized allylamine surfaces by utilizing different time of immersion (2, 6, and 15 h, respectively). Gold nanoparticles are capped with negatively charged carboxylic acid, while plasma surfaces carry positive charge when immersed in a solution of these gold nanoparticles. This leads to electrostatic binding between positively charged AApp surfaces and negatively charged gold nanoparticle surfaces. Later, these samples were washed with Milli-Q water to remove loosely bound nanoparticles and were finally dried with nitrogen.16,19 Cell Culture. Two cell types, the RAW 264.7 (RAW) cells (a murine-derived macrophage cell line) and BMSCs, were applied in this study.2,6 RAW cell cultures were maintained in DMEM (Life Technologies) containing 5% heat-inactivated fetal bovine serum (FBS) (Thermo Scientific) and 1% (v/v) penicillin/streptomycin (Life Technologies) at 37 °C with a 5% CO2 humidified atmosphere. As to the passage procedure, the adherent cells were dislodged by gently passing a cell scraper over the surface of the flask after reaching around 80% confluence, which were then seeded to other flasks. The cells 4503


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Statistical Analysis. All statistical computations were performed using SPSS software, and the statistical significance was analyzed using one-way ANOVA followed by the LSD post hoc test. All the data are shown as means ± standard deviation, and the level of significance was set at P < 0.05.

were expanded through two passages before applied in the subsequent study. BMSCs were isolated and cultured as previously described.2,6,67 Briefly, bone marrow was sourced from patients undergoing elective hip or knee replacement surgery at the Prince Charles Hospital in Brisbane; informed consent was given by all donors, and the ethics approval was from the Ethics Committee of Queensland University of Technology. Mononuclear cells were isolated from the bone marrow by density gradient centrifugation using Lymphoprep (Axis-Shield PoC AS, Oslo, Norway), following the manufacturer’s instructions. The isolated cells were transfered to culture flasks containing culture medium (DMEM supplemented with 10% FBS and 1% (v/v) penicillin/streptomycin) and incubated in a humidified incubator (37 °C, 5% CO2). The unattached hematopoietic cells were removed via medium changes, and the attached cells were passaged using trypsin when they reached 90% confluence. Osteoimmunomodulatory Effects of Controlled Nanotopographies on Macrophages. Cell Morphology and Proliferation. RAW cells were plated on nanoengineered surfaces with different chemistry (AA, AC) and nanotopographies (16, 38, and 68 nm gold nanoparticles) with a density of 104. After overnight growth, lipopolysaccharide (1 μg/mL) was added to activate inflammatory macrophages. After 2 h of stimulation, the media were removed and replaced by serum-free culture medium. After another 6 h, the images of treated cells were captured in a light microscope. Subsequently, the cells were fixed in 4% paraformaldehyde for confocal microscopy and 2.5% glutaraldehyde for SEM and TEM observation, to show growth, spread, and morphology of stimulated macrophages. Inflammatory Response of Macrophages. RAW cells were seeded on different nanoengineered surfaces at a density of 105. The same treatment of LPS (1 μg/mL) was applied to activate inflammatory macrophages. After 6 h, the conditioned media were collected for further BMSC stimulation. Gene expression of inflammatory cytokines (IL-1ra, TNFα, IL-18, IL-1β, and IL-6) and macrophage phenotype markers (CD206, Arginase, CD11c, and CD86) was assessed by RTqPCR. Protein expression of IκB (an inhibitor of NFκB), iNOS, and CD86 (M1 markers) was detected by Western blotting. Activation of Autophagy. The activation of autophagy on macrophages under the same treatment was assayed from both gene and protein levels. Gene expression of autophagy pathway related molecules (Atg5, Atg7, P62, and LC3A/B) was detected by RT-qPCR. Western blotting was carried out to evaluate the protein expression of Atg3, Atg7, P62, LC3, and Becline-1. LC3 fluorescent staining were observed by confocal microscopy. The autophagy ultrastructures were analyzed by TEM. Osteoclastic Activities and Osteogenic Factors. Multinucleated cells were observed by confocal microscopy. The expressions of osteoclastic activity genes [cathepsin K (CTSK), carbonic anhydrase II (CAR2), receptor activator of nuclear factor kappa-B (RANK), MMP9, TRAP, and CR] and osteogenic factor genes (BMP2, BMP6, WNT10b, OSM, VEGF, and TGFβ1) in macrophages under the same treatments were detected by RT-qPCR. Osteogenic Differentiations of BMSCs. To investigate the effect of the nanotopography/macrophage-modulated osteoimmune environment on the osteogenic differentiation of BMSCs, Alizarin Red S staining was used to highlight mineralization nodules in BMSCs grown in a 96-well plate for 14 days in conditioned medium with osteogenic components. The cells were washed with distilled water and fixed in 4% paraformaldehyde for 10 min. After rinsing with distilled water, the cells were stained in a solution of 2% Alizarin Red S at pH 4.1 for 20 min. The samples were air-dried, and images acquired with a light microscope. Quantification detection of the alizarin red staining results was performed following previous protocols.67,68 BMSCs were plated at a density of 1.5 × 105 cells in six-well plates. After incubation for 24 h the culture medium was removed and replaced with conditioned medium followed by 3 days of incubation. Total RNA was harvested and RT-qPCR was performed to detect the gene expression of BMP pathway components (SMAD1, SMAD4, SMAD5, BMPR2, BMPR1a, and BMPR1b) and osteoclastogenesis regulating factors (OPG and RANKL).

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b07808. Detailed experimental procedures; RT-qPCR primers; macrophage morphology under light microscopy, confocal, and SEM; macrophage LC3 fluorescent staining; intracellular autophagy structures observed by TEM; multinucleated cells observed by confocal; morphology of BMSCs under light microscopy; activation of BMP pathway by RT-qPCR; schematic illustration of intracellular response in macrophages (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. Tel: +61 8 83025697. Fax: +61 8 83025689. *E-mail: [email protected]. Tel: +61 7 31386240. Fax: +61 7 31386030. ORCID

Zetao Chen: 0000-0001-8344-2602 Krasimir Vasilev: 0000-0003-3534-4754 Author Contributions #

Z. Chen and A. Bachhuka contributed equally to this paper.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS Funding for this study was provided by the Q-CAS Collaborative Science Fund, The Prince Charles Hospital Foundation, the Osteology Foundation, NHMRC (APP1032738), NHMRC for Fellowship (APP1122825) and ARC (DP120103697 and DP15104212), and the 100 Top Talents Program of Sun Yat-sen University (Z.C.). REFERENCES (1) Chen, Z.; Klein, T.; Murray, R. Z.; Crawford, R.; Chang, J.; Wu, C.; Xiao, Y. Osteoimmunomodulation for the Development of Advanced Bone Biomaterials. Mater. Today 2016, 19, 304−321. (2) Wu, C.; Chen, Z.; Wu, Q.; Yi, D.; Friis, T.; Zheng, X.; Chang, J.; Jiang, X.; Xiao, Y. Clinoenstatite Coatings Have High Bonding Strength, Bioactive Ion Release, and Osteoimmunomodulatory Effects that Enhance in vivo Osseointegration. Biomaterials 2015, 71, 35−47. (3) Wu, C.; Chen, Z.; Yi, D.; Chang, J.; Xiao, Y. Multidirectional Effects of Sr-, Mg-, and Si-containing Bioceramic Coatings with High Bonding Strength on Inflammation, Osteoclastogenesis, and Osteogenesis. ACS Appl. Mater. Interfaces 2014, 6, 4264−4276. (4) Chen, Z.; Yi, D.; Zheng, X.; Chang, J.; Wu, C.; Xiao, Y. Nutrient Element-Based Bioceramic Coatings on Titanium Alloy Stimulating Osteogenesis by Inducing Beneficial Osteoimmmunomodulation. J. Mater. Chem. B 2014, 2, 6030−6043. (5) Chen, Z.; Mao, X.; Tan, L.; Friis, T.; Wu, C.; Crawford, R.; Xiao, Y. Osteoimmunomodulatory Properties of Magnesium Scaffolds Coated with Beta-tricalcium Phosphate. Biomaterials 2014, 35, 8553−8565. (6) Chen, Z.; Yuen, J.; Crawford, R.; Chang, J.; Wu, C.; Xiao, Y. The Effect of Osteoimmunomodulation on the Osteogenic Effects of 4504


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