Magnetic silicium hydroxyapatite nanorods for enhancing osteoblast

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Bio-interactions and Biocompatibility

Magnetic silicium hydroxyapatite nanorods for enhancing osteoblast response in vitro and biointegration in vivo Kai Li, Fang Dai, Ting Yan, Yang Xue, Lan Zhang, and Yong Han ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00073 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 25, 2019

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ACS Biomaterials Science & Engineering

Magnetic silicium hydroxyapatite nanorods for enhancing osteoblast response in vitro and biointegration in vivo Kai Li&, Fang Dai&, Ting Yan, Yang Xue, Lan Zhang*, Yong Han** & co-first author,  Corresponding author, e-mail: [email protected], [email protected], Tel.:+86 02982665580; fax:,+86 02982663453 State-key Laboratory for Mechanical Behavior of Materials, Xi'an Jiaotong University, No.28, Xianning West Road, Xi'an, Shaanxi, 710049, P.R. China Abstract: Osteoblast behavior playing an important role in biointegration of Ti implant with host bone in vivo can be regulated by surface properties and magnetic field. In order to endow Ti surface with good osteogenesis activity, Si mon-substituted and Fe and Si co-substituted hydroxyapatite (HAp) nanorods were fabricated on microporous TiO2 by micro-arc oxidation (MAO) followed with hydrothermal treatment (HT). The surface properties including microstructure, micro-roughness, hydrophilicity, ionreleasing, magnetic property, cytocompatibility and biointegration of substituted HAp nanorods were observed and evaluated, together with pure HAp nanorods and MAOed TiO2 as controls. After doped with Fe, MAOed TiO2 has no changes in phase composition and micro-roughness, whereas it displays weakly ferromagnetic behavior and can enhance osteoblast differentiation in vitro and formation of new bone in vivo, compared with the undoped one. The substituted HAp nanorods adhere firmly to TiO2, and have almost the same wettability and micro-roughness but additional Si, Fe and/or Ca released into medium, compared with pure HAp nanorods. Moreover, the cosubstituted HAp has a small ferromagnetic signal, while its saturation magnetization 1

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value is less than the MAOed doped with Fe. Compared to pure HA nanorods, the substituted HAp nanorods do not only improve cell proliferation and differentiation in vitro, but also enhance ability of bone integration in vivo, especially for the cosubstituted one, which should be ascribed to the combined effect of microstructure, magnetic property and released ions. Keywords: Si and Fe substituted hydroxyapatite; nanorod; magnetism; osteoblast response; biointegration

Introduction Biointegration of Ti implant with host bone is mainly decided by the response of osteogenic-derived cells. Cell microenvironment, especially the properties of implant surface can directly influence cell behaviors (e.g. adhesion, proliferation and differentiation). It is known that natural bone is mainly consisted of nanofibrous collagen and nanosized hydroxyapatite (HAp) doped with Sr2+, Na+, Mg2+, Fe2+, F-, Zn2+, CO32- and silicate etc.1, 2 In order to enhance osteogenesis activity, researchers are interested in designing the microstructure of Ti surface to simulate natural bone. Surface microstructures and properties include nano-topograpghy, phase/chemical composition, wettability, ion releasing and surface energy etc. Their effect on cell behaviors and biointegration has been more or less studied. Recently, surfaces with different nano-topographies (e.g. geometry, diameter, interspace and orientation) have been found to regulate the behavior of cells efficiently, and compared with nanograins, nanorods with special nano-topography can enhance the biointegration with host bone 2


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obviously.3-6 A series of monocrystallized HAp nanorods/nanofibers were designed on Ti to simulate the nano-topography and composition of natural bone in our previous work. It is found that nanorods with a MF>HA≈HS≈HSF. With immersion prolonged, the amount of Ca released from each coating increases, especially for HS and HSF. At 7 d of immersion, Ca amount from HS increases significantly. It is slightly more than those from HSF and CA, and much more than those from MF and HA. At 14 d of immersion, the amount of Ca from each sample further increases, HS also releases the most Ca among the samples, orderly followed by HSF, CA, HA and MF. With further prolonging the immersion time to 28 d, the amount of released Ca is much more from HS than from the others, and follows the order: HS>>HSF>CA>HA>MF. In addition, Si and Fe concentrations from the selected surfaces were evaluated and shown in Figures 5(b) and (c), respectively. With increasing the immersion time, the amounts of Si and Fe from each measured coating increase, and there is no statistic difference of Si amounts between HS and HSF at each immersion time. The trace amount of Fe from MF at each incubation time was detected, indicating Fe was hardly released during the immersion. Whereas, amount of Fe from HSF is much more than that form MF at each immersion time, and its accumulative amount reaches 14.3 ppb after 28 d of immersion. The magnetization curves as a function of the applied magnetic field for MF and HSF were measured with CA as a control. High-field paramagnetic susceptibility has been subtracted from the total hysteretic signal and Figure 5(d) shows independent examination of the ferromagnetic signals of the detected surfaces. With the increased magnetic field, the magnetization of each coating increases rapidly to reach saturation. 18


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The saturation magnetization (Ms) values for CA, MF and HSF are about 0.008, 0.06 and 0.04 emu/cm2, respectively. During the magnetism process, the incorporation of Fe in TiO2 and HAp causes remnant magnetization (Mr) and coercivity (Hc). The inset in Figure 5(d) shows a magnetization curves in the region near H=0 Oe. The values of Mr and Hc for MF are 0.004 emu/cm2 and 43 Oe, respectively, and they are 0.007 emu/cm2 and 70 Oe for HSF. As described in experimental method, when the magnetization behavior of HSF was measured, the detected actually contained the out layer of Fe and Si doped HAp nanorods and the lower layer of Fe-doped TiO2. In order to know the exact magnetization behavior of the nanorods for HSF, a single-layered coating of HAp nanorods doped with similar amounts of Fe and Si was directly prepared on Ti by HT (Figure S5(a)) and its magnetization behavior was measured in Figure S5(b). The magnetization curve (Figure S5(b)) indicates Fe and Si co-doped HAp nanorods have a Ms value of about 0.02 emu/cm2, which is lower than those of MF and HSF. Furthermore, after immersion in PS solution for 14 d, the Ms value of HSF decreases slightly to about 0.034 emu/cm2 (Figure S6), which should be due to the Fe3+ releasing (Figure 5(c)).

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Figure 5(a) Ca, (b) Si and (c) Fe concentrations of the PS solutions immersing the different samples for 1-28 days; (d) the magnetization curves of the coatings of CA, MF and HSF. The other properties including wettability, micro-roughnesses and bonding strength were also evaluated and shown in Figures S7-S9. Compared with Ti, the contact angle is declined to 19.9 ° on CA, and the incorporation of Fe in TiO2 cannot affect the contact angle on MF (Figure S7). After HT, the surfaces with nanorods all show excellent hydrophilcity, and the contact angles are all about 3°for HA, HS and HSF (Figure S7). After HT, the Ra values for HA, HS and HSF slightly decrease to about 2.5 m, but they have not statistic differences, compared with the MAOed ones (Figure S8). The bonding integrity of a coating can be characterized by critical load (Lc) using scratch tests. MAO can fabricate porous TiO2 with high adhesion strength, and even after the fabrication of HAp nanorods by HT, bonding strength of coating is also higher 20


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than the required.38,39 The critical loads of the layers on HS and HSF are both about 34 N, which should be high enough for a coating on implants.39 The delaminations of the initial failures (Marked with squares) occur in the interiors of the layers, further indicating high binding strength of the two layers to Ti (Figure S9). Protein adsorption and osteoblast adhesion and proliferation evaluation in vitro. Figure 6(a) shows the amounts of total proteins on different surfaces from the culture medium after 1 and 24 h of incubation. At each immersion time, compared with Ti, more proteins were adsorbed on MAOed surfaces, and even more on HTed surfaces. With the prolonged immersion time, the amount of protein measured at each surface increase. They are in the following order: HSF>HS>HA>MF>CA>Ti. The mitochondrial activity of osteoblasts on different samples after incubation for different time was shown in Figure 6(b). After incubation for 1 h, compared with Ti, coating surfaces all show higher mitochondrial activity, especially for HA, however, there are no significant diversities among coating surfaces. After 1 day of incubation, mitochondrial activity on all surfaces increases, and it is higher on HTed surfaces compared with that on the MAOed, following the order: HA>HS≈HSF>MF>CA>>Ti. At the incubation of 3 days, the mitochondrial activity on different surfaces increases obviously, indicating the happening of cell proliferation. The mitochondrial activity of cell on HS is the highest, orderly followed by HSF, HA, MF, CA and Ti. With the incubation time prolonged to 7 days, mitochondrial activity on all surfaces further increases, and it follows the trend: HS≈HSF>HA>>MF>CA>Ti.

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Figure 6 (a) total protein adsorbed onto different surfaces after 1 and 24 h of incubation in DMEM containing 10% fetal bovine serum; (b) MTT assays of cell adhesion on different surfaces after 1 h, 1 day and proliferation after 3 and 7 days of incubation. (*) p < 0.05 and (**) p < 0.01 compared with Ti control, (#) p < 0.05 and (##) p < 0.01 compared with CA, (&) p < 0.05 and (&&) p < 0.01 compared with HA. The morphologies of cells seeded on all the coatings after 1 h and 1 d of incubation were observed, as shown in Figure 7 (The left two columns). At incubation for 1 h, cells spread well on the surfaces (The insets in first column), especially on HA, HS and HSF, where many filopodia can be observed at the borders of cells (Marked with red arrows). At 1 d of incubation, cells all show typically polygonal morphologies, and they spread and contact with each other well indicating their good status (Marked with yellow arrows). Furthermore, the viability and distributions of cells on coating surfaces after 1 and 3 days of incubation were evaluated by live/dead staining and shown in the right two columns in Figure 7. At incubation for 1 day, most of the cells were live and stained in green, and few were dead and strained in red, indicating that all the surfaces show high cell viability. With the incubation increased to 3 days, live cells increase obviously on each surface, especially for HA, HS and HSF.

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Figure 7 SEM morphologies of the cells cultured on different surfaces for 1 h and 1 d, the insets showing the lower magnification images (left two columns); fluorescence images showing the distributions and viabilities of the cells cultured on different surfaces for 1 and 3 days (right two columns). In order to further observe the morphologies of adhered cells, vinculin-actin-nucleus tricolors staining fluorescence images of cells on different surfaces after 1 day of culture were taken as shown in Figure 8. The vinculin denoted FA contacts are visible a little in cells on CA and MF, but more obviously in cells on HTed surface. FA contacts are distributed over the peripheral and central regions of cells on HA, HS and HSF, indicating that the cells bind well to them. Compared to the MAOed ones, individual cells on HA, HS and HSF have thicker organized filamentous actin bundles, further 23



indicating their better spread.

Figure 8 Vinculin (green), actin (red), and cell nucleus (blue) fluorescence images of osteoblasts after 24 h of culture on CA, MF, HA, HS and HSF. Osteoblast differentiation evaluation in vitro Differentiation of osteoblasts can be characterized by examining intracellular osteogenesis-related gene expression and intracellular protein content or activity. The expressions of intracellular osteogenesis-related genes, including Runx2, osterix, ALP, OPN, OCN and Col-I were measured and intracellular ALP activity and contents of specific protein (OPN, OCN and Col-I) on different surfaces were evaluated as follows. Osteogenesis-related gene expressions As shown in Figure 9, for Runx2 (Figure 9(a)), Osterix (Figure 9(b)), OPN (Figure 9(d)) and OCN (Figure 9(e)), their expressions at each incubation time on different surfaces generally follow the similar order: HSF>HS>HA>MF>CA, and tend to 24


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increase with the prolonged culturing time. For ALP ((Figure 9(c))), its expression keeps on up-regulating on all the surfaces with incubation prolonged from 3 to 7 days, following the order: HSF>HS>HA>MF>CA. When the incubation time is increased to 14 days, the expression levels of ALP on CA and MF further increase, however, they are down-regulated by HA, HS and HSF compared to those at 7 days of incubation. For Col-I (Figure 9(f)), at 3 d of incubation, its expression on HA is slightly higher than on the other surfaces. At 7 d of incubation, Col-I expresses the most on HSF, the least on CA, and it is almost the same on HA and MF. At 14 d of incubation, on each surface Col-I expression increases obviously, following the order: HSF>HS>HA>MF>CA.

Figure 9 Gene expressions of osteoblasts cultured on different surfaces after incubation for 3, 7 and 14 days: (a) Runx2, (b) Osterix, (c) ALP, (d) OPN, (e) OCN and (f) Col-I. (#) pCA. At 14 days, the ALP activity further increases on CA and MF, whereas decreases on HA, HS and HSF. For the synthesis of intracellular OPN (Figure 10(b)) and OCN (Figure 10(c)), their amounts at each incubation have the same trends: at 3 d of incubation, cells produce relatively the least on CA, while the most on HSF, but there are not significantly statistic differences among MA, HA, HS and HSF; at 7 and 14 d of incubation, their synthesis increases on each surface with prolonging the incubation time, especially on HSF, and they both follow the order: HSF>HS>HA>MF>CA. For Col-I (Figure 10(d)), at each incubation, the

synthesis

amount

on

different

surfaces

is

in

the

following

order:

HSF>HS>HA>MF>CA, and tends to increase on each surface with prolonging the culturing time from 3 to 14 d.

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Figure 10 (a) ALP activity, protein contents of (b) OPN and (c) OCN, and (d) Col-I in osteoblasts cultured on different surfaces after culture for 3-14 days. (#) pCA, and on HA, HS and HSF, cells progress into the period of ECM mineralization as early as incubated for 7 days. Osseointegration of different coatings in vivo Finally, in vivo osseointegration of different coatings was evaluated in rabbits after 8 weeks of implantation and Ti substrates were used as controls. All the samples can induce new bone (NB) formation but with different amount, following the order: HSF ≈HS>HA>MF>CA>Ti (Figure 11(a)). However, the percentage of BIC (Figure 11(b))) and the biomechanical strength of bone-implant integration (Figure 11(c)) both follow a same order: HSF>HS>HA>MF>CA>Ti, suggesting that HSF can accelerate osseointegration in vivo fastest among the coatings herein, which is accordant with the cell responses on the HSF surfaces (Figures 6-10).

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Figure 11 (a) Histological analysis of the implant/bone interfaces after 8 weeks of implantation in the rabbit model, where the tissues stained in red are the newly formed bone and BM is bone marrow cavity, (b) Percentage of bone-to-implant contact (BIC) and (c) pull-out force of different pins after 8 weeks of implantation. Data are presented as means ± SD, n = 4. (*) p < 0.05 and (**) p < 0.01 compared with Ti control, (#) p < 0.05 and (##) p < 0.01 compared with the CA, (&) p < 0.05 and (&&) p < 0.01 compared with the HA.

Discussion Formation the coatings During the MAO process, Ca2+ and Fe3+ from electrolyte penetrated into coating inner by discharge channels,30 and Ca2+ and Fe3+ co-doped TiO2 formed (Figure 1(b)). When MAOed TiO2 was HTed in sodium silicate, based on the formation mechanism 28


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of Sr-doped HAp nanorods6,7 and the results observed (Figures 1-3 and S2-S4), Ca, P and Fe migrated from inner to the outer of TiO2, and they reacted with PO43- and SiO32supplied in HT solution, forming the HAp nuclei, in which Ca and P in HAp lattice were partially replaced by Fe and Si, respectively.6,

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The initial HAp nuclei were

divided into several smaller ones (Figure S4(b)), which could be related with electric neutrality and distortion during the lattice assembling of HAp. With HT process prolonged, HAp nuclei grew rapidly in length and became nanorods (Figures S4(c) and (d)). After the substitution of P by Si (HS), crystal cell of HAp had a small expansion compared with HA (Figure S2), however, it contracts after additional substitution of Ca by Fe ions, which should be due to the mismatching of ion sizes. The lattice distortion of HS and HSF increases the Ca releasing (Figure 5). It is noticed that Ca releases more from HS than from HSF during the immersion, and it may be due to the decreased amount of Ca (Figure 1) and the contraction of HAp lattice of HSF (Figure S2). Moreover, due to a high concentration of SiO32- in HT solution, SiO32- not only participated in the formation of HAp, but also reacted with Ti4+, Ca2+ and OH-, forming CST nanoplates,39 as shown in Figures 3 and S4. The nanoplates are partly crystallized (Figure 3) and their lattice assembling should be more slowly than the crystallized HAp nanorods, and it results in a gradual covering of CST nanoplates by HAp nanrods (Figure S4(d)). Magnetic properties of MF and HSF It has been confirmed by experiment and theoretical calculation that Fe-doped anatase and rutile have magnetism.40, 41 Although the reported are powder and mainly 29



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fabricated by wet chemistry technique, the theory of combined interaction of oxygen vacancies and magnetic cation impurities to explore their magnetic properties40,

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should be also appropriate for the Fe3+ doped TiO2 coating herein (Figure 5(d)). Fe3+ ions substitute Ti4+ sites in TiO2, leading to the creation of oxygen vacancies (to maintain charge neutrality). The electron and Vo overlaps with the d shells, and bound magnetic polarons (Fe3+-VO-Fe3+) form, endowing MF with magnetism according to Fcenter exchange mechanism.40 Regarding Fe substituted HAp, Jiang et al reported the local geometry and distribution of Fe2+ and Fe3+ in HAp lattice as early as 2002, finding that in the HA, Fe3+ preferentially occupied Ca (1) positions.27, 42 The substitution of Ca2+ by Fe3+ induces a transition from diamagnetic to paramagnetic, and endows HAp nanorods on HSF with magnetic property (Figures 5(d) and S5). Compared with MF, Ms of HSF decreased and it should due to the low magnetism of outlayer of HAp nanorods (Figure S5) and a decreased magnetism of the inner layer of TiO2 which contains less Fe related to that before HT process. 43 The amount of Fe incorporated in HAp affects its magnetic property.27 After immersion in PS solution, when the Fe ions were consistently released from HSF (Figure 5(c)), the magnetic property of HAp should be getting weaker. Fortunately, Ms value of HSF slightly decreases to 0.034 emu/cm2 after immersion for 14 d (Figure S6), indicating that the whole magnetic property of HSF does not reduced obviously. A series work about the change of magnetic property of HAp after long-time immersion and the effect of change on the cell response will be continued in the future. Cell response in vitro and biointegration in vivo 30


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Once an implant is inserted in vivo to repair bone defect, proteins initially adsorb on its surface, followed by adhesion, proliferation and differentiation of osteogenesis related cells. Remodeling process of new bone around the implant is mainly decided by cell response, which is directly related with surface properties.44 There are no significant statistic difference of cell adhesion and proliferation between CA and MF (Figure 6(b)). The differentiation of osteoblasts characterized by osteogenesis-related markers (Figure 9) and intracellular proteins or activity (Figure 10) in vitro, and osteogensis ability evaluated by new bone formation in vivo (Figure 11) indicate that compared with CA, MF can facilitate the osteogenic differentiation and biointergation with host bone. CA and MF have the similar surface morphology, phase composition, hydrophilcity and roughness, but MF has lower content of Ca2+, additional Fe3+ and magnetic behavior, compared with CA. The amount of Fe released from MF is hardly to be detected, about 3 ppb after 28 d of immersion (Figures 5(c)), indicating its negligible effect on cell response. Additional Ca ions can facilitate osteogenesis differentiation of osteoblast,45 whereas, the Ca released from MF is less than that from CA (Figure 5(a)). So, the additionally magnetic behavior of MF should be in charge of the enhanced differentiation of hFOB1.19 and biointegration in vivo. According to Zablotskii et al,22-24 ion-channel on/off switching events as well as cell membrane potential, which regulate cell functions (e.g. cell division, cell reprogramming, differentiation), can be affected by a magnetic field with high gradient value. For example, a magnetic field with a gradient value on the order of 108–109 Tm-1 can directly change the cell membrane potential by 1-10 mV. Magnetic nanoparticles can 31



induce a high-gradient magnetic field due to the nanosize,24 inducing the change in value of membrane potential and accelerated differentiation of hFOB1.19 on MF. Compared with the MAOed, cell response in vitro (Figures 6-10) and biointegration in vivo (Figure 11) were enhanced obviously on HTed samples. The outlayers of HTed surfaces are HAp in chemical composition which are more bioactive than TiO2.46 They are more hydrophilic (Figure S7) and can absorb more protein than the MAOed surfaces (Figure 6(a)). The type, amount and conformation of the adsorbed protein play an important role in cell binding events on the material surface. Nanorods and hydrophilic surface are reported to adsorb fibronectin (FN) and vitronectin (VN) efficiently from serum, efficiently.46-49 FN and VN are the anchoring proteins required for integrinreceptor based cell adhesion47-49, and more hydrophilic surfaces leads the adsorbed VN and FN to be in a more bioactive conformation for cell adhesion.47,48 So, cells adhered and spreaded better on HTed surfaces than on the MAOed (Figure 8). For different cell response on HT-formed nanorods, HA consists HAp nanorods with an interrod-spacing of 70 nm, which has been proved to have a significantly improved cell response in vitro and bone-integration in vivo compared with CA in our previous work 6, 9, and its cell response and bone integration are also confirmed here (Figures 811). HS has almost the same phase composition and hydrophilcity with HA, but different nanotopography and additional Ca and Si releasing (Figures 1 and 5). Compared with HA, mitochondria activity on HS is slightly lower that than on HA during the cell adhesion process (Figure 6(b)), which may due to the effect of interrod spacing on cell response6, 9. However, Figure 8 shows HS can support focal adhesion 32


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formation well which is similar to HA after 1d of incubation. Furthermore, HS shows enhanced proliferation and differentiation of hFOB1.19 and biointegration in vivo, compared with HA (Figure 6-11). The substitution of P by Si distorts the lattice of HAp, resulting in additional Si as well as more Ca released to the culture medium (Figures S2, 5(a) and 5(b)). Extracellular Ca2+ increase induces an increase of intracellular Ca2+ in the osteoblast through voltage gated Ca2+ channels, and it enhances cell proliferation, differentiation and mineralization.45 Simultaneously, Si could be bound to glycosaminoglycan and plays an important role in forming cross-links between collagen and proteoglycan, and it is an initiator of osteoblast mineralization.15 So compared with HA, the negative effect on osteoblast behavior and bone-integration induced by the interrod spacing of HS should be overwhelmed by the positive impact of the released Ca and Si (Figures 8-11). Compared with HS, HSF has the similar phase composition, hydrophilcity, nanotopography and Si concentration released into medium. However, the substitution of Ca by Fe in HSF results in the decreased Ca but additional Fe releasing and magnetic property (Figure 5). Compared with HS, HSF has almost the same cell adhesion and proliferation (Figures 6-10), but facilitated osteoblast differentiation, which is characterized by osteogenesis-related markers at level of genes (Figure 9), intracellular proteins or activity (Figure 10), and biointegration in vivo (Figure 11). It is known that iron is necessary to facilitate the assembly of functional Fe-S cluster proteins and ribonucleotide reductases, and it plays an important role in cell cycle progression.50, 51 As described in the experimental method, the culture medium was refreshed every other 33



day. Although after immersion from 14 to 28 d, Fe3+ releasing decreased obviously and the accumulated concentration was little (about 4 ppb), its accumulated concentration was about 1-2 ppb every two days at the initial immersion time (e.g. 1-14 d). Zhang et al found that consistent supplementation of Fe3+ in a concentration range of 1.5 to 12.7 ppb during incubation for more than 7 days (e.g. 10 or 14 days) could promote differentiation of osteogenesis-derived cells.50 So, the released Fe3+ should contribute to the enhanced cell response on HSF during the culture period from 1 to 14 days. Based on the effect of micro-magnetic field on cell response and new bone formation of MF, magnetic property of HSF also should contribute to its enhanced cell behaviors and biointegration. Although it cannot be concluded here that whether released Fe3+ or magnetic property of HSF plays the dominate role in the HSF performance, their combined effect is obvious. By analyzing the cytocompatibility and osteogenesis activity of different coatings, compared to the MAOed TiO2 (CA), the combined effect of nanotopography, chemical composition (HAp), releasing of Ca, Si and Fe as well as magnetism endows HSF with an obviously enhanced cell response in vitro and bone integration in vivo.

Conclusion Fe and Si co-substituted HAp nanorods were fabricated on microporous TiO2 by a hybrid process of microarc oxidation and hydrothermal treatment. During the HT process, Ca, Fe and P migrated from TiO2, and reacted with OH-, PO43- and SiO32- in HT solution, forming HAp nanorods. In HAp lattice, partial Ca and P were substituted 34


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by Fe and Si, respectively. Fe doped TiO2 has no changes in phase composition and micro-roughness, whereas shows weakly ferromagnetic behavior, and enhanced cell differentiation and bone integration, compared with undoped one. Si and Fe cosubstituted HAp has a lower Ms value compared with MF, whereas it has more improved cell proliferation, differentiation and new bone formation than Si monosubstituted HAp nanorods, even more than pure HAp nanorods. The combined effect of its microstructure, magnetic property and released ions of Fe, Ca and Si should contribute the enhanced cell response in vitro and biointegration in vivo.

Supporting information Schematic diagram showing the rectangle-marked region for histological analysis of the coated pillar implanted in rabbit femur for 8 weeks; an enlarged view of XRD pattern in the 26-35°of 2θ for HA, HS and HSF; cross-section morphologies and element distributions of different samples; surface morphologies of the MAO coating after hydrothermal treatment for different times and schematic diagram showing nucleation and growth of HSF; surface morphology and magnetization curve of the Fe and Si co-doped HA which was directly fabricated on Ti substrate; contact angles of different surfaces; the laser confocal images and average Ra value of different samples; scratch morphologies of the HS, HSF coatings, together with the amplified views of the areas of marked initial failure caused by Lc; magnetization curve of the HSF coating after immersed in PS solution for 14 days. Conflict of interest 35



There are no conflicts to declare. Acknowledgements We appreciate Research Fund for the National Natural Science Foundation of China (Grant number 51771142, 51571158, 51631007)), National Key Research and Development Program of China No. 2016YFC1100600 (sub-project 2016YFC1100604) for financially supporting this work. References (1) Olszta, M.J.; Cheng, X.; Jee, S. S.; Kumar, R.; Kim, Y.; Kaufman, M. J.; Douglas, E. P.; Gower, L.B. Bone structure and formation: A new perspective, Mater. Sci. Eng. R 2007, 58, 77-116. DOI:10.1016/j.mser.2007.05.001 (2) Bigi, A.; Cojazzi, G.; Panzavolta, S.; Ripamonti, A.; Roveri, N.; Romanello, M.; Suarez, K. N.; Moro, L. Chemical and Structural Characterization of the Mineral Phase from Cortical and Trabecular Bone, J. Inorg. Biochem.1997, 68, 45-51. DOI:10.1016/S0162-0134(97)00007-X (3) Roure, O.; Saez, A.; Buguin, A.; R.H. Austin, Philippe Chavrier, Pascal Silberzan and Benoit Ladoux. Force mapping in epithelial cell migration, PNAS, 2005, 102, 23902395. DOI: 10.1073/pnas.0408482102 (4) Fu, X.; Xu, M.; Liu, J.; Qi, Y.; Li, S.; Wang, H. Regulation of migratory activity of human keratinocytes by topography of multiscale collagen-containing nanofibrous matrices, Biomater. 2014, 35, 1496-1506. DOI:10.1016/j.biomaterials.2013.11.013 (5) Xie, J.; Macewan, M.R.; Ray, W.Z.; Liu, W.; Siewe, D.Y.; Xia, Y. Radially aligned, electrospun nanofibers as dural substitutes for wound closure and tissue regeneration 36


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Table of Contents graphic

Magnetic silicium hydroxyapatite nanorods for enhancing osteoblast response in vitro and biointegration in vivo Kai Li&, Fang Dai&, Ting Yan, Yang Xue, Lan Zhang*, Yong Han** & co-first author,  Corresponding author, e-mail: [email protected], [email protected], Tel.:+86 02982665580; fax:,+86 02982663453 State-key Laboratory for Mechanical Behavior of Materials, Xi'an Jiaotong University, No.28, Xianning West Road, Xi'an, Shaanxi, 710049, P.R. China 44


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Si and Fe co-substituted hydroxyapatite nanorods enhance osteoblast response in vitro and biointegration in vivo due to the combined effect of nanotopography, chemical composition, magnetism and ion-releasing.

45



Figure 1 Surface morphologies of different samples: (a) CA, (b) MF, (c) HA, (d) HS and (e) HSF, the tables inserted are the corresponding element contents; (f) XRD patterns of different samples. 101x47mm (300 x 300 DPI)


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Figure 2 TEM images of scratched nanorod from the surfaces of HS (a, b, c) and HSF (d, e, f): (a) and (d) bright-field images; (b) and (e) EDS patterns taken from the area marked with a ring in (a) and (d), respectively; (e) and (f) HRTEM of the nanorods marked with a ring in (a) and (d), respectively; insets in (a) and (d) showing the corresponding SAED, respectively. 101x99mm (300 x 300 DPI)



Figure 3 TEM images of scratched nanoplates from the surface of HSF: (a) bright-field image, (b) EDS pattern of the area marked with a ring in (a), (c) SAED pattern taken from the area marked with a ring in (a), and (d) HRTEM of the nanoplate marked with a ring in (a). 101x102mm (300 x 300 DPI)


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Figure 4 XPS patterns of the different samples: (a) survey, (b) Fe2p and (c) Si2p; (d) FTIR spectrums of different surfaces. 101x83mm (300 x 300 DPI)



Figure 5 (a) Ca, (b) Si and (c) Fe concentrations of the PS solutions immersing the different samples for 128 days; (d) the magnetization curves of the coatings of CA, MF and HSF. 101x84mm (600 x 600 DPI)


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Figure 6 (a) total protein adsorbed onto different surfaces after 1 and 24 h of incubation in DMEM containing 10% fetal bovine serum; (b) MTT assays of cell adhesion on different surfaces after 1 h, 1 day and proliferation after 3 and 7 days of incubation. (*) p < 0.05 and (**) p < 0.01 compared with Ti control, (#) p < 0.05 and (##) p < 0.01 compared with CA, (&) p < 0.05 and (&&) p < 0.01 compared with HA. 101x37mm (300 x 300 DPI)



Figure 7 SEM morphologies of the cells cultured on different surfaces for 1 h and 1 d, the insets showing the corresponding magnified images (left two columns); fluorescence images showing the distributions and viabilities of the cells cultured on different surfaces for 1 and 3 days (right two columns). 203x179mm (300 x 300 DPI)


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Figure 8 Vinculin (green), actin (red), and cell nucleus (blue) fluorescence images of osteoblasts after 24 h of culture on CA, MF, HA, HS and HSF. 203x123mm (300 x 300 DPI)



Figure 9 Gene expressions of osteoblasts cultured on different surfaces after incubation for 3, 7 and 14 days: (a) Runx2, (b) Osterix, (c) ALP, (d) OPN, (e) OCN and (f) Col-I. (#) p

Magnetic silicium hydroxyapatite nanorods for enhancing osteoblast

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