Surface Modification by Divalent Main-Group-Elemental Ions for

Article Cite This: ACS Biomater. Sci. Eng. 2019, 5, 3311−3324

pubs.acs.org/journal/abseba

Surface Modification by Divalent Main-Group-Elemental Ions for Improved Bone Remodeling To Instruct Implant Biofabrication Jiaxing Gong,†,‡,#,⊥ Miao Sun,†,#,⊥ Shaolong Wang,†,# Jianxiang He,†,# Yu Wang,†,# Ying Qian,†,# Yu Liu,†,# Lingqing Dong,†,#,§ Liang Ma,∥ Kui Cheng,§ Wenjian Weng,§ Mengfei Yu,*,†,# Yu Shrike Zhang,*,‡ and Huiming Wang*,†,# †

The Affiliated Stomatologic Hospital, School of Medicine, Zhejiang University, 395 Yanan Road, Hangzhou 310003, China Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 65 Landsdowne Street, Cambridge, Massachusetts 02139, United States # Key Laboratory of Oral Biomedical Research of Zhejiang Province, 268 Kaixuan Road, Hangzhou 310029, China § School of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Zhejiang University, 38 Zheda Road, Hangzhou 310027, China ∥ State Key Laboratory of Fluid Power & Mechatronic Systems, School of Mechanical Engineering, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China

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ABSTRACT: Divalent main-group-elemental ions are widely used to improve osteogenic capacity of implants biofabricated from Ti and its alloys. However, the conclusions regarding their osseointegration and immunogenicity are always inconsistent because of the multiple bone remodeling processes as well as the distinct material surface features arising from processing. Here we successfully manufactured the porous micro/nanostructured surface topography with divalent main-groupelemental ions (Mg2+, Ca2+, Sr2+, Ba2+) on substrates through hydrothermal treatment and comprehensively evaluated the complex bone remodeling processes, including osseointegration, immunogenicity, and fibrosis of substrates and implants. We found that Sr-modified implants not only upregulated the adhesion and proliferation of mesenchymal stem cells but also the differentiation of osteogenic markers compared with those modified by other divalent main-group-elemental ions (Mg2+, Ca2+, Ba2+). More importantly, the osteoclastogenesis, immunogenicity, and fibrosis of Sr-modified implants were also significantly downregulated. In vivo, evaluations of new bone formation and histological morphology at the interface of implant and host as well as the removal torque similarly indicated the improved osseointegration of Sr-modified implants as well as the absence of immunogenicity, fibrosis, or necrosis. Our results suggested that among various divalent main-group-elemental ions, Sr2+ might be a promising one for enhancing bone remodeling, which can be used to instruct functionalization of the surfaces of biofabricated Ti-based orthopedic and dental implants in the future. KEYWORDS: implant, bone remodeling, osseointegration, osteoclastogenesis, immunogenicity, divalent main-group-elemental ions

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after implantation.11,12 The polarization of macrophages to pro-inflammatory phenotype or anti-inflammatory phenotype can affect osseointegration in different ways.13,14 Moreover, fibrous tissues might intervene at the interface between the host and the embedded implant.15,16 To promote osseointegration of implants, numerous studies have focused on the modification of Ti surfaces using divalent main-group-elemental ions. Mg is the fourth most-abundant element in the body and essential for bone metabolism in all stages.17 Yoshizawa18 and Wu19 found that Mg can stimulate

INTRODUCTION

Ti and its alloys have been widely used for dental implants and prosthesis components because of their excellent biocompatibility and mechanical properties, corrosion resistance, and amenability to biofabrication in custom shapes and structures.1−4 However, early osseointegration between the host bone and the implants is still insufficient.5,6 Bone remodeling and stability after implantation are influenced by multiple factors. First, surrounding bone marrow-derived mesenchymal stem cells (BMSCs) with osteogenic differentiation potential could generate osteoblasts and form new bone.7,8 Second, an immune reaction, a process termed osteoclast/osteoblast “coupling”,9,10 is considered as a vital component in determining biomaterial-mediated bone tissue reconstruction © 2019 American Chemical Society

Received: February 22, 2019 Accepted: May 23, 2019 Published: May 23, 2019 3311

DOI: 10.1021/acsbiomaterials.9b00270 ACS Biomater. Sci. Eng. 2019, 5, 3311−3324

Article

ACS Biomaterials Science & Engineering

Figure 1. (A) Sample preparation procedure. (B) SEM images of surface features and (C) EDS analyses of elemental distributions of Mg-, Ca-, Sr-, and Ba-modified and pristine Ti substrates. Scale bar = 5 μm.

body, several studies have demonstrated that it can obviously upregulate osteoblastic differentiation, as demonstrated by an increment of hydroxyapatite deposition.26−28 However, conclusions of previous studies are always inconsistent, partly because of the lack of systematic investigations of osseointegration, immunogenicity, and fibrosis.15,16 In this study, we successfully manufactured the porous micro/nanostructured surface topography with divalent maingroup-elemental ions (Mg2+, Ca2+, Sr2+, Ba2+) on substrates through hydrothermal treatment and comprehensively evaluated the bone remodeling processes both in vitro and in vivo. The similar porous hierarchical micro/nanostructured surface topography was achieved after processing with different ions using hydrothermal treatment in various ionic solutions. The possible mechanism underlying the superior osseointegration as well as the reduced immunogenicity and fibrosis of Srmodified implants was also discussed. Our findings may provide insights into surface functionalization of biofabricated Ti-based orthopedic and dental implants in the future.

new bone formation through extracellular matrix (ECM) proteins and transcription factors. Moreover, Mg is responsible for cell adhesion through interactions with integrin receptors of osteoblasts.20 Ca is the most abundant metal element in our body, and the existence of calcium phosphates can form appropriate local environments for promoting the functions of different tissues.21 Insertion of Ca2+ and Mg2+ into the interlayers of sodium titanate nanostructures was previously found to effectively enhance protein adsorption and thus promote the adhesion of rat BMSCs and bone mineralization.22 Implants containing Ca can also induce bone formation while reducing bone resorption.19,23 Sr is also an element of group IIA that is close to Ca physically. It can accelerate the proliferation and osteogenesis of osteoblasts by activating the ERK-MAPK and Wnt signaling pathways, together with the apoptosis of osteoclasts through a calcium-sensing receptor (CaSR)-dependent mechanism.24 When coated on the surface of implants and slowly released into the local environment, it can inhibit bone resorption and promote bone formation.16,25 Although Ba is not an essential trace element in the human 3312


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Figure 2. (A) Morphological observation of BMSCs on Mg-, Ca-, Sr-, and Ba-modified and pristine Ti substrates. SEM images show that the cells adopted different morphologies on different substrates. Scale bars = (left) 10 and (right) 5 μm for the Mg-modified, Ca-modified, and Ti groups and (left) 20 and (right) 10 μm for the Sr- and Ba-modified groups. (B) Focal adhesion point and cytoskeleton immunofluorescence staining of BMSCs on Mg-, Ca-, Sr-, and Ba-modified and pristine Ti substrates. Scale bars = 50 μm. BMSCs were stained for F-actin (red), vinculin (green), and nuclei (blue). (C−E) Corresponding quantitative analyses of the maximum Feret diameter (C), cytoplasm−nuclear ratio (D), and cell area (E) on different substrates (n > 50; *, p < 0.05; **, p < 0.01).

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RESULTS AND DISCUSSION Surface Characterizations of the Substrates. The surface topography of these substrates after hydrothermal treatment (Figure 1A) was characterized by scanning electronic microscopy (SEM). The similar porous hierarchical micro/nanostructured surface topographies with respective phases of MgTiO3, CaTiO3, SrTiO3, BaTiO3, and TiO2 were achieved after hydrothermal treatment (Figure 1B), and the mean pore diameters were 166.72 ± 133.29, 170.72 ± 66.47, 179.65 ± 77.88, 212.84 ± 151.61, and 179.77 ± 160.68 nm, for the Mg-, Ca-, Sr-, and Ba-modified and pristine Ti substrates, respectively. Several studies had proven that micro/nanoscale topography could enhance osteogenic differentiation.26,29 The energy-dispersive X-ray spectrometry (EDS) results further indicated that all of the elements (Mg, Ca, Sr, Ba) were distributed uniformly (Figure 1C). The surface element contents were measured to be 0.49% for Mg, 2.98% for Ca, 0.51% for Sr, and 3.35% for Ba. Evaluation of Osteointegration of Various Implants. Cellular adhesion behaviors on different substrates were observed by SEM and confocal laser scanning microscopy (CLSM), as shown in Figure 2A and Figure 2B, respectively. On the pristine Ti surface, the BMSCs poorly spread, lacking pseudopodium extensions and cellular propagation fronts. Contrarily, the cells on Mg-, Ca-, and Ba-modified Ti surfaces exhibited more elongation with more prominent pseudopodia, while BMSCs anchored to the Sr-modified substrates presented the largest cellular areas and exhibited interconnected and more apparent lamellipodia. CLSM images also confirmed that BMSCs on the Sr- and Ba-modified surfaces displayed more visible pseudopodia (Figure 2B), which

demonstrated higher vinculin (green) expression than cells on other surfaces. Staining of the cytoskeleton (red) proved that the cellular areas were evidently larger and betterdeveloped on the Sr- and Ba-modified surfaces, whereas BMSCs on the other substrates remained relatively small. Quantitative analyses of the Feret diameter (Figure 2C), cytoplasm−nuclear ratio (Figure 2D), and cellular area (Figure 2E) also revealed improved cell spreading behavior on the Srmodified surfaces, followed by those modified with Ba2+, compared with the other substrates (p < 0.01 or p < 0.05). Live/dead staining and the Cell Counting Kit-8 (CCK-8) assay were used to determine cell proliferation. Figure 3D shows a comparison of cell densities on different substrates after 1, 3, and 5 days of culture. There was no significant difference among the groups at 1 day of culture. However, on the third day, number of cells on the Sr-modified substrates turned out to be higher than those on others (p < 0.05 or p < 0.01). The trend was significantly amplified on day 5, when BMSCs on the Sr-modified substrates were 20% higher in number than those in other groups (p < 0.01). These results were further confirmed by the live/dead staining (Figure 3A), where the quantitative analysis showed that the number of BMSCs on the Sr-modified substrates was significantly higher than the others on both days 3 and 5 (p < 0.01), as revealed in Figure 3B. Moreover, the cell areas on Sr-modified substrates were also larger than those in other groups (p < 0.01 or p < 0.05; Figure 3C). We further used real-time quantitative polymerase chain reaction (RT-qPCR) to analyze the gene expression levels associated with the osteogenic-differentiation-related markers (Col-1, RunX-2, and OCN) of BMSCs after 7 and 14 days of culture in the osteoinductive medium.27 As demonstrated in 3313


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Figure 3. (A) Viability of BMSCs on Mg-, Ca-, Sr-, and Ba-modified and pristine Ti substrates after 1, 3, and 5 days of culture, as demonstrated by live/dead staining (live cells, green; dead cells, red). Scale bar = 500 μm. (B, C) Corresponding quantitative analyses of (B) average cell numbers and (C) average cell areas. (D) Cellular viability analyses (CCK-8 assay) of BMSCs on Mg-, Ca-, Sr-, and Ba-modified and pristine Ti substrates after 1, 3, and 5 days of culture. *, p < 0.05; **, p < 0.01.

of cytokines such as interleukin (IL)-6 and IL-8 secreted by these macrophages.28 As indicated in Figure 4B, the expressions of all osteogenic genes in the Sr-modified group were still the highest among all of the substrates on both day 7 and day 14 (p < 0.05 or p < 0.01). Increased expressions of RunX-2 and OCN were also observed in the Ba-modified samples (p < 0.05 or p < 0.01), whereas the expression levels in the Ti group consistently ranked the lowest (p < 0.05 or p < 0.01). Previous research found that Mg-, Ca-, Sr-, or Ba-substituted or -coated bioactive glasses, scaffolds, or implants could elicit a promotive effect on osteogenesis through sustained existence of divalent main-group-elemental ions.30,31 Our research also confirmed that divalent cations of Sr2+ seemed to play an important role in cell adhesion, proliferation, and osteogenesis. First, BMSCs anchored to the Sr-modified substrates exhibited larger cellular ranges and good extent of morphological elongation with more prominent pseudopodia than cells on other substrates. The CLSM images further demonstrated that

Figure 4A, when cultured in the osteoinductive medium, cells on the Sr-modified substrates expressed more Col-I, RunX-2, and OCN than other groups at both 7 and 14 days (p < 0.01). In the remaining groups, the expression levels of Col-I, RunX2, and OCN for cells in the Ba-modified group were higher than those on Mg- and Ca-modified surfaces as well as pristine Ti substrates at 14 days of culture (p < 0.05 or p < 0.01). The fact that the results we gained from RT-qPCR were relative rather than absolute values should have minimized the influence of the osteoinductive medium used in these in vitro studies. The exception to this trend was that the expressions of Col-1 and RunX-2 on the Ti substrates ranked second and were followed by Ba-, Mg-, and Ca-modified substrates at 7 days of culture. These results showed that osteogenic differentiation of BMSCs on the Sr-modified substrates was significantly more pronounced than on the other surfaces. We further evaluated the osteogenic differentiation of BMSCs in conditioned medium collected from 24 h of culture of RAW264.7 cells, which contained several types 3314


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Figure 4. (A, B) RT-qPCR analyses of relative gene expressions of osteogenic differentiation markers (Col-1, RunX-2, and OCN) on Mg-, Ca-, Sr-, and Ba-modified and pristine Ti substrates after 7 and 14 days of culture in (A) the normal osteoblast-inducing medium and (b) the conditioned osteoblast-inducing medium (after 24 h of culturing with RAW264.7). All of the data were normalized to gene expressions of the corresponding markers at day 7. (C, D) RT-qPCR analyses of relative gene expressions of (C) M2-related markers (Arg-1, IL-10, and TGF-β) and (D) M1-related markers (CD86, TNF-α, and iNOS) on Mg-, Ca-, Sr-, and Ba-modified and pristine Ti substrates after 4 and 6 days of culture. *, p < 0.05; **, p < 0.01.

the cells on Sr-modified substrates had higher vinculin expression. Second, the number of BMSCs on the Sr-modified substrates was significantly higher than others after 3 and 5 days of culture. It is further noteworthy that the Sr coating induced higher expressions of osteogenic differentiation markers (Col-I, RunX-2, and OCN) of BMSCs, which indicated that the cells on Sr-modified substrates expressed more osteogenic-associated genes and entered into the stage of osteogenic differentiation after 7 or 14 days of culture. Moreover, the results of osteogenic differentiation in conditioned medium suggested that the effect of inflammatory

cytokines could have been restrained by the coatings, especially the Sr2+ ion. The Sr element likely plays an important role in bone remodeling through activation of the CaSR, and it may increase ERK1/2 phosphorylation, thus promoting osteoblast replication.32 Osteoclastogenesis Evaluation of Various Implants. Apart from the osteogenic differentiation potential, immune reactions after implantation in vivo are considered as a vital component in determining biomaterial-mediated bone tissue reconstruction.33 It was reported previously that Sr- and Mgcoated implants showed higher osteoclastogenesis-inhibiting 3315


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Figure 5. (A) Morphological observation of osteoclasts on Mg-, Ca-, Sr-, and Ba-modified and pristine Ti substrates after 6 days of culture of RAW264.7 cells, stained for F-actin (red) and nuclei (blue). Scale bar = 200 μm (left columns) and 100 μm (right columns). (B, C) Corresponding quantitative analyses of (B) relative cell numbers and (C) relative cell areas on different substrates (n > 50; *, p < 0.05; **, p < 0.01).

macrophages is an essential part of the immunomodulatory property of biomaterials. The polarization switch between classically activated M1 phenotypes and alternatively activated M2 phenotypes occurs in response to the microenvironment, which can have positive or negative impacts on osteogenic differentiation through the production of cytokines and chemokines. M1 macrophages can secrete pro-inflammatory factors (iNOS, TNF-α, and IL-1A) that strengthen osteoclastic activities and contribute to bone resorption; in contrast, M2 macrophages are recognized to express anti-inflammatory factors (Arg-1, IL-10, and TGF-β) that benefit bone healing and regeneration.38 However, studies concerning the immunomodulatory function of Ti substrates coated with divalent main-group elements are still insufficient. To probe the effects of coated substrates on the polarization of macrophages, we analyzed the gene expression levels of M1-related markers (CD86, TNF-α, and iNOS) and M2-related markers (Arg-1, IL-10, and TGF-β) of RAW264.7 macrophages after 4 and 6 days of culture. Figure 4C demonstrates not only that Srmodified surfaces increased Arg-1 expression on day 4 (p < 0.05 or p < 0.01) but also that the expressions of IL-10 and TGF-β on day 6 were significantly upregulated (p < 0.01 or p < 0.05), followed by Ca-, Ba-, and Mg-modified substrates. Macrophages in the pristine Ti group expressed lowest

capacity than hydroxyapatite, which demonstrated that these elements might have higher biological stability.8 The effects of strontium ranelate on women with postmenopausal osteoporosis also confirmed that Sr can reduce the risk of fracture by inhibiting the activity of osteoclasts and reducing the rate of bone remodeling.34 In our study, RAW264.7 cells started to be induced to osteoclasts under the effect of receptor activator of nuclear factor-κB ligand (RANKL) for 4 days. As shown in Figure 5A, after 6 days the number of osteoclasts (defined as cells containing three or more nuclei; shown in blue) on Ti substrates was the highest, and furthermore, their sizes (red) were also the largest, followed by Mg- and Ca-modified substrates. Sr-modified surfaces had the smallest and fewest osteoclasts compared with the other substrates (p < 0.01; Figure 5B,C). We speculated that in osteoclasts, Sr could sensitize the CaSR and elicit osteoclast apoptosis through activation of the PLC, Akt, and NF-κB pathways, subsequently modulating gene expression and cell apoptosis.8,35 Macrophage Polarization on Various Implants. In a medication-related osteonecrosis of the jaw (MRONJ) model, it was found that zoledronic acid could exacerbate inflammation through M1 macrophage polarization.36 Some other studies confirmed that M2 macrophage polarization accelerates bone mineralization.37 We infer that the polarization of 3316


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Figure 6. (A) Viability of NIH/3T3 fibroblasts on Mg-, Ca-, Sr-, and Ba-modified and pristine Ti substrates after 1, 3, and 5 days of culture, as demonstrated by live/dead staining (live cells, green; dead cells, red). Scale bar = 500 μm. (B, C) Corresponding quantitative analyses of (B) average cell numbers and (C) average cell areas. (D) Cellular viability analysis (CCK-8 assay) of NIH/3T3 fibroblasts on Mg-, Ca-, Sr-, and Bamodified and pristine Ti substrates after 1, 3, and 5 days of culture. *, p < 0.05; **, p < 0.01.

amounts of M2-related markers (p < 0.01 or p < 0.05). Moreover, Figure 4D shows that the cells cultured on Srmodified substrates expressed the lowest amount of either CD86, TNF-α, or iNOS (p < 0.01 or p < 0.05), followed by the Ba-modified group. As expected, the Ti group had the opposite phenomenon, where levels of M1-related markers were much higher than those in the Sr- and Ba-modified groups (p < 0.01 or p < 0.05). As revealed by these RT-qPCR results, substrates coated with Sr generally inhibited macrophage switching to classically activated M1 phenotypes and promoted their activation to the alternative M2 phenotypes, thus further reducing the inflammatory response and associated osteoclastic giant cell response (Figure 5A). Fibrosis Evaluation of Various Implants. The host fibroblasts from the periodontium always attach to an implant prior to osteoblasts, and the former easily form fibrous capsules at the tissue−implant interfaces as a typical wound healing response due to the foreign body reactions. These capsule can

interfere the stability and other long-term functions of the implants.39,40 At 24 h after seeding of the NIH/3T3 fibroblasts, their viability was examined. The overall viability was high in all groups (Figure 6A). The number of NIH/3T3 fibroblasts in the Ti group was significantly higher than those on other substrates after 3 and 5 days of culture. The numbers of cells on the Sr- and Ba-modified substrates were the fewest (p < 0.05 or p < 0.01), as shown in Figure 6A,B. Moreover, the cell areas on Sr-modified substrates were also smaller than those in other groups (p < 0.01 or p < 0.05; Figure 6C). These results were further demonstrated by the CCK-8 assay, which showed a comparison of NIH/3T3 fibroblast numbers on different substrates after 1, 3, and 5 days of culture (Figure 6D). After 1 day of culture, the numbers of NIH/3T3 fibroblasts on the Srand Ba-modified substrates appeared to be less than others (p < 0.05 or p < 0.01). This trend was amplified on days 3 and 5. Meanwhile, the cells on the Mg-modified substrates appeared similar to those on the Ti group and ranked second. Moreover, 3317


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Figure 7. (A) Mineralization apposition by fluorescence labeling of alizarin red S (red with star) and calcein (green with arrow) for the Sr-modified, Ba-modified, CI, and Ti groups after 4 and 8 weeks of implantation. Scale bar = 800 μm. (B) Corresponding quantitative analysis of MARs (μm/ day) (n = 4; *, p < 0.05; **, p < 0.01). (C) 3D reconstruction images showing osteogenesis of the Sr-modified, Ba-modified, CI, and Ti groups in vivo examined by micro-CT analyses after 4 and 8 weeks of implantation. Bone, gray; implants, green. (D, E) Qualitative analyses of osteogenesis at 4 and 8 weeks after implantation. The indices include (D) BV/TV and (E) BMD (n = 4; *, p < 0.05; **, p < 0.01).

the amount of total collagen can usually be used to indicate fibrotic activity.41 After 14 days of culture, the secretions of collagen and total proteins by the NIH/3T3 fibroblasts were further evaluated. The proportions of collagen to total proteins in Mg-modified substrates and Ti were slightly higher than those in the Ca-, Sr-, and Ba-modified groups (p < 0.05). Figure S2 also shows that quantifications of collagen secretions were similar for all five metal bases. In Vivo Tests. Here we chose the groups that performed relatively better in vitro (Sr- and Ba-modified) for the animal experiments, as well as the pristine Ti control group. In addition, we took clinically used implants (CIs) (produced by ZDI, GuangCi, China) as the standard control. The Ti implants were cleaned ultrasonically and then soaked in 10 M NaOH solution at 120 °C for 2 h. After they were cooled and washed, the implants were steeped in 0.1 M HCl for 1 h. Later, the implants were steeped in saturated SrCl2 or BaCl2 solution and deionized water for 12 h at 37 °C. After annealing at 600 °C for 1 h, the Sr- and Ba-modified implants along with the control Ti implants were obtained. After operation, we used sequential fluorescence labeling to measure the new-bone mineralization apposition rates (MARs, in μm/day) at days 10 to 18 (4-week group) and days 38 to 46 (8-week group) postimplantation (the timeline and schematic diagram are

illustrated in Figure S1). Fluorescence labeling was formed along the implanted Ti thread (Figure 7A: (red) alizarin red, 18 days before the animals were sacrificed; (green) calcein, 10 days before animals were sacrificed). Moreover, the quantitative statistics results presented in Figure 7B revealed that MARs in the Sr- and Ba-modified groups were similar (1.82 ± 0.55 and 1.77 ± 0.44 μm/day, respectively, for the 4-week group and 1.83 ± 0.55 and 1.68 ± 0.69 μm/day, respectively, for the 8-week group; p > 0.05). The MARs in the CI and Ti groups (1.38 ± 0.37 and 1.25 ± 0.45 μm/day, respectively, for the 4-week group and 1.29 ± 0.46 and 1.14 ± 0.42 μm/day, respectively, for the 8-week group) were lower than those in the Sr- and Ba-modified groups (p > 0.05). Microcomputed tomography (micro-CT) scans were used to demonstrate the bone healing parameters. Figure 7C shows the bone morphology (gray) around the implants (green) through 3D reconstruction images. After 4 or 8 weeks of operation, implants and bones integrated well in all of the groups, especially the Sr-modified surface, which was covered with the greatest amount of bone tissue. Figure 7D shows the corresponding result that the bone volume/total volume (BV/TV) values for the modified implants (Sr 4-week, Sr 8week and Ba 4-week, Ba 8-week) were larger than those for the control CI and Ti implants (p < 0.01). In Figure 7E, Sr 4-week 3318


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Figure 8. (A) Histological morphologies of methylene blue/acid fuchsin staining showing the bone (red with star) forming around the Ti implants (black) and the bone-to-implant contact (arrow) in the Sr-modified, Ba-modified, CI, and Ti groups after 4 and 8 weeks of implantation. Scale bars = 500 μm in the left column and 200 μm in the right column. (B, C) Histomorphometrical analyses of (B) BIC% and (C) BA% around the threads at 4 and 8 weeks (n = 4). (D) Removal torque values for the Sr-modified, Ba-modified, CI, and Ti groups after implantation for 4 and 8 weeks (n = 6). *, p < 0.05; **, p < 0.01.

directly contacting the implant surface in the hard-tissue slicing to the entire length of the implant. The BIC% in the Sr 4-week group was 63.33 ± 6.56%, which was significantly higher than the values of 49.71 ± 4.29%, 45.49 ± 8.50%, and 40.55 ± 7.38% in the Ba 4-week, CI 4-week, and Ti 4-week groups, respectively (p < 0.05 or p < 0.01; Figure 8B). In addition, bone area ratio (BA%) was calculated as the area percentage of bone tissue with respect to the entire area around the implanted thread. The BA% in the Sr 4-week group (59.38 ± 3.51%) was much higher than those in the Ba 4-week, Ca 4week, and Ti 4-week groups (53.85 ± 1.67%, 51.90 ± 2.52%, and 43.41 ± 2.52%, respectively) (p < 0.05 or p < 0.01; Figure 8C). At 8 weeks postimplantation, more new bone formed, with the presence of normal bone marrow and Haversian canal. Figure 8A also clearly reveals that the Sr 8-week and Ba 8-week implant threads were in direct contact with the surrounding bone tissues with minimum fibrous capsule formation at the interfaces. These observations indicated that the Sr- and Bamodified implants did not promote the growth of fibroblasts

and Sr 8-week implants had the highest bone mineral density (BMD) values compared with the other groups at the same time point (p < 0.01 or p < 0.05). These results indicated that new bone mineralized well in the early stages postimplantation and formed faster and higher in volume in the Sr-modified and Ba-modified groups compared with the CI and Ti groups. These results also indicated that significant amounts of bone were formed without any additional osteoinductive cues but the implants themselves. Furthermore, histological analysis was performed to evaluate the new bone formation and bone−implant interfaces. In the methylene blue/acid fuchsin staining images, the bones, fibrous tissues/cartilage, and implants are indicated in red, white/blue, and black, respectively (Figure 8A). At 4 weeks after surgery, no obvious necrosis or inflammation occurred in any group, and new bones formed well around the implanted threads. The Sr 4-week group formed more new trabecular bone, followed by the Ba 4-week group. Bone-to-implant contact (BIC%) was used to compare the distance of the bone 3319


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Figure 9. (A) HE staining of Sr-modified, Ba-modified, CI, and Ti groups after decalcification. Bone trabecular, BT; blood vessels, star. (B) TRAP staining showing purple osteoclasts (arrow). Scale bars = 500 μm for A1, B1, A2, and B3 and 200 μm for B2 and B4.

± 0.04 N m) (p > 0.05; Figure 8D). However, at 8 weeks postsurgery, the removal torque continued rising to 0.50 ± 0.03 N m in the Sr 8-week group, which was significantly higher than those in other groups (Ba 8-week, 0.36 ± 0.02; Ca 8-week, 0.33 ± 0.02; Ti 8-week, 0.34 ± 0.05) (p < 0.01). These results of animal tests echoed our in vitro findings that the existence of Sr on implant surfaces might best promote bone healing among divalent main-group-elemental ions. Sr is a natural bone trace element whose concentration is typically very low but nevertheless can activate the CaSR when it reaches a certain concentration range. The CaSR was initially reported to be expressed in cells of the osteoblast lineage in vitro42−46 and in the skeleton.47,48 To date it has been known that CaSR is a physiological regulator in bone cell metabolism.32 Some studies have suggested that activation of the CaSR results in increasing replication in osteoblastic cells via ERK1/2 signaling32 as well as the prevention of osteoblast apoptosis through the CaSR by activation of Akt.21 Moreover, investigations also found that the CaSR may play an important role in modulating osteoclast behaviors through control of gene expressions and cell survival,49 i.e., increasing the apoptosis of osteoclasts through activation of PLC and NFκB. Although there is evidence that the effect of Sr is mediated by cation-sensing receptors other than the CaSR,32,50 this standpoint has been refuted by other studies, which have demonstrated that the effect of Sr is CaSR-dependent.51,52 Considerable literature indicates that Sr can bind to and activate the CaSR as a full agonist, resulting in activation of PLC, ERK1/2, and Akt in vitro.53−55 In vivo, the physiological content of Sr is low and thus cannot activate the CaSR under natural conditions.39 When coated on the surfaces of implants and slowly released into the local environment, it could form a high concentration at the implantation sites, therefore increasing proliferation and osteogenesis of osteoblasts as well as apoptosis of osteoclasts. However, these hypotheses regarding signaling pathways induced by Sr in our particular

on the interfaces and benefited early bone integration, consistent with the results of fibrosis evaluation in vitro (Figure 6). The BIC% in the Sr 8-week group increased to 77.07 ± 8.80%, while those in the Ba 8-week, CI 8-week, and Ti 8-week groups increased only to 59.34 ± 2.78%, 50.32 ± 4.74%, and 49.69 ± 11.92%, respectively, suggesting that the Sr 8-week group had the highest BIC% and that Ba 8-week group ranked second, both of which were significantly higher than those in the CI 8-week and Ti 8-week groups (p < 0.01 or p < 0.05; Figure 8B). In addition, the BA% of the Sr 8-week group (69.73 ± 1.51%) also ascended compared with the Sr 4-week group and presented much higher values than those in the Ba 8-week, CI 8-week, and Ti 8-week groups (60.33 ± 1.52%, 57.43.90 ± 1.41%, 53.67 ± 2.52%, respectively) (p < 0.01; Figure 8C). As shown in Figure 9A1,A2, no obvious inflammatory response or necrosis was observed, and there were large numbers of bone trabecular (BT) with vascular formation and blood cells inside (black stars). In the TRAP-stained sections (Figure 9B1−B4), purple osteoclasts (red arrows) with three or more nuclei could be found at the edges of newly formed bone matrices. Larger numbers of osteoclasts were stained in the CI and Ti groups compared with the Sr and Ba groups. These results indicated that divalent cations Sr2+ and Ba2+ had an inhibitory effect in osteoclast maturation, even better than the CI, and the effect of Sr2+ was most significant. These results were consistent with the osteoclastogenesis evaluation in vitro (Figure 5), where Sr-modified surfaces were shown to present the smallest and fewest osteoclasts, with a strong inhibitory effect on osteoclast maturation. Moreover, removal torque testing was performed to evaluate the interfacial shear strength between the implants and the surrounding bones. The torque when the implants were initially inserted was approximately 0.15 N m. After 4 weeks, the torque, which reflects the maximum osseointegration of the implants, all increased, especially in the Sr 4-week group (0.32 3320


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detached with 0.25% trypsin and 1 mM EGTA and passaged when the cells grew to 70−80% confluence. Cell Viability Analysis. The BMSCs were seeded on the substrates placed in the wells of 24-well plates at a density of 1 × 104 cells/well. After culturing for 1, 3, and 5 days, cell attachment and proliferation were assessed using the CCK-8 assay. In brief, at the designed time points, the substrates were transferred to the wells of new 24-well plates and rinsed with phosphate-buffered saline (PBS) (Gibco). Then 500 μL of α-MEM and 50 μL of the CCK-8 solution (Dojindo Laboratories) were added to each well, followed by incubation at 37 °C for 2 h. Finally, the absorbance was measured at 450 nm with a Sunrise-Basic spectrophotometer (TECAN, Switzerland). The viability analysis of NIH/3T3 fibroblasts was carried out in the same way, except that α-MEM was replaced by DMEM with high glucose. Live/Dead Staining of Cells. The BMSCs and NIH/3T3 fibroblasts were seeded on the substrates in a 24-well plate at a density of 1 × 104 cells/well. After culturing for 1, 3, and 5 days, specimens with cells were rinsed with PBS and incubated with 2 mg/ mL calcein-AM (Invitrogen, USA) and 1 mg/mL ethidium homodimer-1 (Invitrogen) for 30 min. The samples were then rinsed with PBS and visualized by inverted fluorescence microscopy (Carl Zeiss, Germany). Morphological Observation of BMSCs. To evaluate the early adhesion of BMSCs on the different substrates, the cell morphology was studied by SEM. The BMSCs were seeded on the substrates placed in 24-well plates at a density of 1 × 104 cells/well, and after culturing for 18 h, the samples were fixed in 2.5% glutaraldehyde overnight at 4 °C. After that, the samples were dehydrated first with alcohol at concentrations of 30%, 50%, 70%, 80%, 90%, 95%, and 100% and then in a Hitachi model HCP-2 critical point dryer using liquid CO2. After the BMSCs were coated with gold−palladium, their morphologies were observed by SEM. Immunofluorescence Staining of BMSCs. The BMSCs were seeded on the substrates placed in 24-well plates at a density of 1 × 104 cells/well. After culturing for 18 h, the samples were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.05% Triton X100, and then blocked with 2% FBS and 2% bovine serum albumin (BSA) (Sigma-Aldrich, USA) in PBS. The primary antibodies used were the mouse antivinculin polyclonal antibody (ab18058, Abcam) and rhodamine-phalloidin (PHDR1, Cytoskeleton, USA). The secondary antibody was the fluorescein isothiocyanate (FITC)conjugated antimouse secondary antibody (A-11001, Thermo Fisher Scientific, USA), and the nuclei were counterstained with 4′,6diamidino-2-phenylindole (DAPI) (H-1200, VECTOR, USA). The samples were visualized by CLSM (Nikon A1R/A1, Japan). Real-Time Quantitative Polymerase Chain Reaction Analysis. RT-qPCR was used to detect the expression levels of osteogenicdifferentiation-related genes (collagen 1 (COL-1), osteocalcin (OCN), and runt-related transcription factor 2 (RunX-2)) of BMSCs in different samples. The cells were seeded on the substrates at a density of 5 × 104 cells/well and cultured in osteoblast-inducing medium for 7 or 14 days. Osteoblast-inducing medium consisted of αMEM with 10% FBS, 10 mmol/L β-glycerol phosphate (SigmaAldrich), 0.1 μmol/L dexamethasone (Sigma-Aldrich), and 0.2 mmol/L ascorbic acid (Sigma-Aldrich). To verify the osteogenic differentiation ability under inflammation conditions, we also mixed osteoblast-inducing medium (α-MEM medium base) and DMEM (with high glucose) that had been cultured with RAW264.7 cells for 1 day at a ratio of 1:1. Total RNA was extracted using TRIzol reagent (Invitrogen). The total RNA concentration was determined using a NanoDrop 2000c instrument. An equivalent amount of RNA from each sample was reverse-transcribed into complementary DNA (cDNA) using the Superscript II first-strand cDNA synthesis kit (TaKaRa, Japan). The primers for the target genes are listed in Table S1. RT-qPCR analysis of genes was performed on an Applied Biosystems 7500 system using SYBR Premix Ex Taq (TaKaRa). Forty cycles were used to amplify all of the gene sequences, and the comparative expression levels were obtained by transforming the logarithmic values into absolute values using 2−ΔΔCT. The expression

implants are still premature and would require systematic investigations and validations in the future.

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CONCLUSIONS In this study, we demonstrated that modification with divalent main-group-elemental ions (Mg2+, Ca2+, Sr2+, Ba2+) is able to positively mediate bone remodeling on Ti-based implant surfaces. They can not only upregulate the adhesion, proliferation, and osteogenic differentiation of BMSCs but also downregulate osteoclastogenesis, inflammatory reactions, and fibrosis. Considering the in vitro and in vivo experimental results, we suggest that the use of Sr-modified implants might be a promising strategy to achieve improved bone remodeling for orthopedic and dental applications and may also be conveniently applied to a wide variety of biofabricated implants such as those produced through 3D printing.

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MATERIALS AND METHODS

Sample Preparation. Ti (99.99% purity, Hongshengti, China) with a thickness of 1 mm was processed into pieces of 1.0 cm × 1.0 cm in dimension as substrates. These substrates were polished with SiC abrasive papers of small grain size (#1500−4000) and then cleaned ultrasonically in ethanol and deionized water. NaOH (analytical reagent (AR), SCR, China) was dissolved in deionized water to a final concentration of 10 M, and this solution containing the substrates was sealed in a Teflon-lined stainless steel autoclave and maintained at 120 °C for 2 h. After cooling to ambient temperature, the Ti substrates were taken out and washed three times in deionized water. Subsequently, the substrates were steeped in 0.1 M HCl (38%, SCR) for 1 h. After being washed three times, the substrates were steeped in saturated MgCl2, CaCl2, SrCl2, or BaCl2 (AR, SCR) solution and deionized water, respectively, for 12 h at 37 °C. The substrates were later washed and annealed at 600 °C for 1 h. UV treatment was carried out using a 15 W bactericidal lamp (Toshiba, Japan) at room temperature for 30 min before use. The flowchart is shown in Figure 1A. The surface topographies of the Mg-, Ca-, Sr-, and Ba-modified and pristine Ti surfaces were examined by SEM using a Hitachi model TM-1000 scanning electron microscope. The mean pore diameters of the porosity were measured by Image Pro Plus (Media Cybernetics, USA). The elemental compositions of these substrates, contents, and mappings of Mg, Ca, Sr, and Ba were determined by EDS on a Hitachi instrument. Culture of BMSCs, RAW264.7 Cells, and NIH/3T3 Fibroblasts. BMSCs were isolated and collected from Sprague-Dawley (SD) rats. Briefly, 3-week-aged male SD rats were sacrificed by overdose of anesthetics. It was shown before that the quantity and properties of BMSCs derived from 3- to 6-week-old rats are both highly suited for osteogenesis studies.56 Bone marrow was collected from the femurs and tibiae, suspended in a growth medium containing α-modified Eagle’s medium (α-MEM) (Gibco, USA) and 10% fetal bovine serum (FBS) (Sciencell, USA), and cultured in a humidified atmosphere of 5% CO2 at 37 °C. Nonadherent cells were removed by changing the medium after 1 day, and the culture medium was changed every 2 days thereafter. For subculture, the cells were detached with 0.25% trypsin (Amresco, USA) and 1-mM ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (Amresco, USA) and were passaged when the cells grew to 70−80% confluence. All experiments with BMSCs were conducted with cultures at passage 3. RAW264.7 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) with high glucose containing 10% FBS and incubated at 37 °C under 5% CO2. For subculture, the cells were detached by pipet blowing. NIH/3T3 mouse embryo fibroblasts were obtained from American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 15% FBS. Cells were incubated at 37 °C under 5% CO2. For subculture, the cells were 3321


Article

ACS Biomaterials Science & Engineering levels of the target genes were normalized to that of the housekeeping gene GAPDH. RT-qPCR was also used to detect the expression levels of M1/M2 macrophage-related genes (CD86, tumor necrosis factor α (TNF-α), inducible nitric oxide synthase (iNOS), arginase 1 (Arg-1), interleukin 10 (IL-10), and transforming growth factor β (TGF-β)) of RAW264.7 in different samples. Osteoclast Differentiation of RAW264.7 Cells. RAW264.7 cells were seeded on substances in 24-well plates at a density of 1 × 104 cells/well in the presence of 50 ng/mL RANKL (NOVUS, USA). The culture medium was changed every 2 days. After 6 days, the substrates were fixed in 4% paraformaldehyde for 15 min in room temperature, permeabilized with 0.05% Triton X-100 in PBS, blocked with 2% FBS and 2% BSA in PBS, and stained with rhodaminephalloidin. The nuclei were counterstained DAPI. The samples were visualized by an inverted fluorescence microscope (Carl Zeiss). Cells with three or more nuclei were defined as multinucleated giant cells, or osteoclasts. Collagen Assay and Total Protein Detection. The total collagen in NIH/3T3 fibroblasts was detected by Sirius Red staining, and the total protein was measured by Fast Green staining. NIH/3T3 fibroblasts were seeded on different substances in 24-well plates at a density of 1 × 104 cells/well, and the culture medium was changed every 2 days. After culturing for 14 days, the cells were fixed in Bouin’s fluid (Solarbio, China) for 1 h at room temperature. After being washed three times and dried, the cells were incubated in Sirius Red dye containing 0.04% Fast Green (Solarbio) and 0.1% Sirius Red F3B (Solarbio) for 1 h. The dye in the supernatant was washed off using ddH2O, and 0.1 M NaOH was used to dissolve the dye for 30 min on a shaker. Finally, 80 μL of stop solution (Solarbio) was added to each well in a 96-well plate to terminate the reaction, and the plate was shaken for another minute. The resulting optical density (OD) values at 450 nm represented the content of collagen, and the OD values at 630 nm represented the total protein. Animals and Surgical Procedure. Twenty-four New Zealand rabbits (male, weight 2500−3000 g) were used for experiment. The experiment was approved by the Animal Care and Ethics Committee of Zhejiang Academy of Medical Sciences (2018-096). Animals were randomly chosen with different insertions at different times (4 and 8 weeks): Sr-coated implants (Sr 4-week and Sr 8-week), Ba-coated implants (Ba 4-week and Ba 8-week), Ti implants (Ti 4-week and Ti 8-week, Control I), and clinical used implants (CI 4-week and CI 8week, Control II, produced by ZDI, GuangCi, China). Thus, each animal carried four implants, two in the distal end of the femur and two in proximal end of tibia (per time point Sr-/Ba-/CI/Ti, n = 12). Anesthesia (0.3 g/mL pentobarbital sodium; Merck, USA) was given to the rabbits intravenously (1.0 mL per kg). After shaving and disinfection, local anesthesia was performed with primacaine (4% articaine hydrochloride and 1/100000 adrenaline; Pierre Rolland) by subcutaneous injection. Then a 20 mm incision was made, and the tibia and femur were both exposed. The implant holes were prepared using a start drill, 2.2 mm drill, 3.3 mm drill, and profile drill (ZDI, GuangCi, China). The implant position is presented in Figure S1, and the torsion was about 0.15 N m. Soft tissue was closured with a 4-0 suture (Ethicon, Johnson & Johnson Medical GmbH, Norderstedt, Germany). After the operation, 10 units/kg penicillin (Beyotime Biotechnology, China) was injected intramuscularly for 3 days (once daily) to prevent the rabbits from infection. At 18 days before sacrifice, 20 mg/kg alizarin red (Sigma-Aldrich) was injected intramuscularly, and 8 days later 10 mg/kg calcein (Sigma-Aldrich) was injected in the same way. Twelve of the 24 rabbits were sacrificed 4 weeks after operation, and the remaining animals were terminated at 8 weeks. Histomorphometric Analysis. The distal femurs with implants (n = 4 per time point) were fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich) for 24 h and then penetrated and embedded by neutral resins. A high-speed precision microtome (Leica 2500E) was used to cut the samples and ground sections containing the central part and parallel to the long axis of the implants; after polishing, the final thickness of the slice was approximately 50 μm. The slice was stained with methylene blue and acid fuchsin. Bone-to-implant

contact (BIC%) was measured to compare the length of the bone directly contacting the implant surface to the entire length of the implant in the bone. The bone area ratio (BA%) was calculated as the area percentage of bone tissue relative to the entire area of thread (0.6 mm around the implant). The mineralization apposition rate (MAR, μm/day), representing the average rate over 8 days (the duration between alizarin red and calcein injections), was measured using the distance between the red and green fluorescent strips. Images and data were obtained and analyzed using a DM4000 bright-field microscope (Leica, Germany) and Image Pro Plus (Media Cybernetics, USA). Removal Torque Testing. The dissected tibiae with implants (n = 6 per time point) were used in removal torque testing to evaluate implant stability in the bone. The removal torque value (RTV) in Newton centimeters (NC) reflects the interfacial shear strength between the implant and the surrounding bone. The peak RTV was measured by an electronic torsion testing machine (digital MGT-12 torque meter, Mark-10 Corporation, USA), and the compression speed was 5 deg/min. Microcomputed Tomography. Femurs with implants (n = 6 per time point) were collected from rabbits 4 and 8 weeks postoperation, and the bones were dissected, cleaned, fixed in 10% PFA at room temperature for 2 days, and transferred to 100% ethanol. Scans were performed using a voxel size of 8.96 μm, a scanning resolution of 18 μm, an operating voltage of 45 kV, a current of 500 μA, and a rotation step of 0.6° (180° of angular range) on a Bruker micro-CT system. The NRecon software (Micro Photonics, Belgium) was used for 3D reconstruction and viewing of images. After 3D reconstruction, the CTvox software (Bruker micro-CT) was used for bone analysis. The bone mineral density (BMD) and directly measured bone volume fraction (bone volume/total volume (BV/TV)) were calculated. As shown in Figure S3, BMD and BV/TV were measured from the first thread of the implant to where the last thread disappeared. The diameter of the measurement was 3.7 mm, leaving a 0.2 mm border of the formed bone layer, from which the calculations were derived. The thresholds were set in the range of 50−80. Immunohistochemistry. Immunohistochemistry was performed (n = 2 per time point) to detect bone formation, vascularization (hematoxylin and eosin (HE) staining), and osteoclasts (TRAP staining). The dissected femurs with implants were fixed in 4% PFA solution and decalcified in 10% ethylenediaminetetraacetic acid (EDTA) (SCR) for approximately 1 month. The implant was removed gently later and the bone block was embedded in paraffin. The slice was approximately 4 μm in thickness. Statistical Analysis. All values are expressed as mean ± standard deviation. Statistical analysis was carried out by a one-way analysis of variance, and all statistical analyses were performed using the Prism software. p values less than 0.05 were considered statistically significant (*, p < 0.05; **, p < 0.01).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.9b00270. Timeline and schematic diagram of the animal experiment, figure showing the proportions of collagen to total proteins and representative Sirius Red staining images, schematic diagram showing micro-CT measurements of BMD and BV/TV, and a table showing primer sequences used for RT-qPCR (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (M.Y.). *E-mail: [email protected] (Y.S.Z.). *E-mail: [email protected] (H.W.). 3322


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Lingqing Dong: 0000-0002-2203-3212 Kui Cheng: 0000-0003-4828-6450 Wenjian Weng: 0000-0002-9373-7284 Mengfei Yu: 0000-0002-7700-4697 Yu Shrike Zhang: 0000-0002-0045-0808 Author Contributions ⊥

J.G. and M.S. contributed equally to this work. J.G. and M.Y. conceived and designed the experiments. J.G. and M.S. carried out the experiments. M.S., S.W., J.H., Y.W., Y.Q., Y.L., L.D., K.C., W.W., L.M., Y.S.Z., and H.W. analyzed the data. J.G., Y.S.Z., and M.Y. wrote the manuscript. All of the authors revised the manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51502262, 81600838, 81670972), the Key Research and Development Program of Zhejiang, China (2017C01054, 2018C03062), the Medical Technology and Education of Zhejiang Province of China (2016KYB178, 2018KY501), the Research and Development Program of Hangzhou, Zhejiang (20171226Y50), the Zhejiang Provincial Chinese Medical Science Research Foundation (2016ZB077), the P o stdo ctoral S cience F oundation of China (2017M621923), and Zhejiang University Education Foundation Global Partnership Fund.

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