Strontium-substituted Nanohydroxyapatite Coatings to Decrease

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Applications of Polymer, Composite, and Coating Materials

Strontium-substituted Nanohydroxyapatite Coatings to Decrease Aseptic Loosening of Femoral Prosthesis: Safety and Efficacy in a Rat Model Genwen Mao, Chuang Wang, Qian Li, Tonghe Zhu, Jianhai Yang, Tao Fu, and Xing Ma ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b16758 • Publication Date (Web): 14 Feb 2019 Downloaded from http://pubs.acs.org on February 15, 2019

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ACS Applied Materials & Interfaces

Strontium-substituted Nanohydroxyapatite Coatings to Decrease Aseptic Loosening of Femoral Prosthesis: Safety and Efficacy in a Rat Model Genwen Mao1,2,#, Chuang Wang3,#, Qian Li4, Tonghe Zhu5,6, Jianhai Yang7, Tao Fu8,**, Xing Ma1,2,* 1

Department of Orthopedics, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, Shaanxi, PR China. 2 Center of Joint Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, Shaanxi, PR China. 3 Department of Orthopedics, The Ninth Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710054, Shaanxi, PR China. 4 Department of Orthopedics, The First Hospital of Shijiazhuang, Shijiazhuang 050011, Hebei, PR China. 5 Department of Sports Medicine, The Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai 200233, PR China. 6 State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Chemistry, Chemical Engineering and Biotechnology of Donghua University, Shanghai 201620, PR China. 7 School of Aerospace, Xi’an Jiaotong University, Xi’an 710049, Shaanxi, PR China. 8 Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, Shaanxi, PR China. *Corresponding author, E-mail address: [email protected] ** Corresponding author, E-mail address: [email protected] # Authors contributed equally. Keywords: Nanohydroxyapatite, strontium, plasma spraying, hip replacement, aseptic loosening of prosthesis Abstract This study aimed to evaluate the safety and efficacy of strontium-substituted nanohydroxyapatite (Sr-nHA) coatings in decreasing aseptic loosening of the femoral prosthetic stem after hip replacement surgery in a rat model. Sr-nHAs (atomic ratio of Sr/(Ca + Sr) of 0%, 5%, 10% and 20%) were prepared by homogeneous phase coprecipitation. Plasma spraying was employed to prepare the Sr-nHA coatings on titanium rod substrate. After microstructure analyses, we investigated the safety and efficacy of Sr-nHA coatings both in vitro and in vivo. Cytotoxicity, histocompatibility, general toxicity, mineralization and osteogenic differentiation tests demonstrated that the Sr-nHA coatings had good safety and efficacy in vitro. Histological observation, fluorescence microscopy, microcomputed tomography, biomechanical test and energy dispersive of X-ray analysis revealed that the Sr-nHA coatings were safe and effective

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in vivo. In both in vitro and in vivo experiments, the efficacy of the Sr-nHA coatings revealed a dose-dependent effect and the 20% Sr-nHA coating was more effective than the other three coatings. Employing Sr-nHA coatings on titanium implants for surface modification showed safety and great application potential in decreasing the aseptic loosening of femoral prostheses after hip replacement. This kind of coating provides a method for decreasing aseptic loosening of femoral prostheses.

Introduction Hip replacement is the most effective method to treat bone and joint destruction caused by severe bone and joint degenerative diseases, joint damage and other diseases1,2. Total hip arthroplasty (THA), which offers an excellent and effective treatment for patients with hip pain or dysfunction, is lauded as “the operation of the century” by the Lancet3. According to the US Agency for Healthcare Research and Quality, at least 285,000 patients undergo THA every year in the United States, and more than 600,000 patients undergo total knee arthroplasty4. With the broadening of surgical indications and the improvement of prosthetic materials for THA5-7, the number of patients who have developed hip dysplasia and have received THA is steadily increasing8-10. The number of surgeries for hip replacement is predicted to double twice in the next 20 years11. Despite the continuous development of artificial joint materials and the maturity of joint replacement surgery, a certain number of patients experience failure due to various complications after joint replacement, which ultimately leads to another joint replacement surgery and requires joint revision surgery12,13. At present, the common causes of failure of artificial joint replacement are aseptic loosening of the prosthesis, infection, stress shielding, prosthetic micromotion, periprosthetic fracture and pain14. Aseptic loosening of the prosthesis is the most important and common long-term complication after artificial joint replacement. According to the annual report of the UK National Joint Registry, from April 1, 2003, to December 31, 2015, the total number of hip revisions and knee revisions in the UK due to aseptic loosening of the prosthesis was 38,310 and 16,711 cases, respectively15. Therefore, how to prevent aseptic loosening of the prosthesis and prolong the life of artificial joints is an important issue that many medical workers have been studying. The mechanism is still unclear. Generally, osteolysis is believed to be related to the debris generated from the joint surface, which induces inflammatory reactions in the


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tissue surrounding the prosthesis and promotes the excessive formation of osteoclasts and bone resorption; this formation then triggers osteolysis and pseudomembrane formation around the prosthesis and finally results in prosthesis loosening16-20. Therefore, inhibiting the osteolysis process could prevent prosthetic loosening16-21. The most used biomaterial for decreasing aseptic loosening in clinical practice is HA coating applied to the implants. HA deposition on the surface of implants enhances the interaction between cells and medical implant by enhancing adhesion, proliferation

and

osteogenic

differentiation

of

osteoblast

cells,

with

osseous-integration promoted22, 23. However, aseptic loosening of femoral prosthesis remains common12-15. Therefore, more studies are still needed on decreasing aseptic loosening. Strontium promotes bone mesenchymal stem cells osteogenic differentiation, osteoblasts proliferation and inhibits osteoclast proliferation, which together promote bone regeneration and inhibit bone resorption, being a treatment used for osteoporosis in clinics worldwide. Many studies have reported that when strontium is used to partially replace calcium in HA, this replacement, which creates Sr-nHA, not only changes the dissolution kinetics and improves biodegradability but also has better bone formation, biocompatibility and osteoinductive capacity than that of the undoped HA24-28. As we discuss above, osteolysis mediated by osteoclasts is the key aspect in aseptic loosening. Therefore, strontium substitution may theoretically be a choice for decreasing aseptic loosening by inhibiting osteolysis and improving the bone-forming properties. In this study, we deposited 0%, 5%, 10% and 20% Sr-nHA powders on the surface of titanium rods by plasma spraying technique. The coated titanium rods as a model of the femoral prosthetic stem were inserted into the femoral cavity of rats to explore the safety and efficacy of Sr-nHA coatings for decreasing aseptic loosening. We hypothesized that strontium-substituted nanoHA might provide a safe and efficacious means to decrease aseptic loosening of the femoral prosthesis.

Materials and Method Sr-nHA powders preparation The 0%, 5%, 10% and 20% Sr-nHAs (Figure 1A) were prepared by a one-step method, which mainly used the principle of homogeneous phase coprecipitation25, 29-33. The Sr-nHAs were designed with the Sr/(Ca + Sr) atomic ratios of 5%, 10% and 20%.



The details were published in the literatures29-33. Briefly, the undoped nanoHA and Sr-nHA powders were prepared based on the reaction as follows: (10 - X) CaCl2 + X SrCl2 + 6 Na3PO4 + 2 NaOH = (Ca10-

x

Srx) (PO4)6(OH)2 + 20 NaCl. Taking 10%

Sr-nHA for example, all reagents used were analytical grade chemicals (CaCl2, SrCl2 and Na3PO4 powders were from Junsei Chemical Co., Ltd., Tokyo). First, the solution containing CaCl2 (0.36 mol/L) and SrCl2 (0.04 mol/L) and the solution Na3PO4 (0.24 mol/L) were prepared with ultrapure water. The atomic ratio of Sr/(Ca + Sr) was set as 10%. Second, the two solutions with equal volume were mixed and the mixture was continuously stirred at 37 °C. The pH value was adjusted to 10 by adding 1 mol/L of NaOH solution dropwise34, 35. The reaction continued under stirring at 37 °C for 2 h. Finally, white precipitation was washed with ultrapure water by centrifugation at least 5 times. The slurry was dried in an oven at 120 °C for 3 days and ground to fine powders. Sr-nHA coating preparation The prepared powders (Figure 1A) were coated on titanium rods (diameter 1.0 mm, Figure 1B) with a plasma spray equipment (Sulzer Metco, METCO-9M, Switzerland). The spray parameters were as follows: argon plasma gas flow rate of 80-90 cubic foot per minute (cfpm), hydrogen plasma gas flow rate 17.5-20 cfpm, spray distance 90-100 mm, current 480 A, voltage 70 V, powder feeding rate 50 g/min and maximum power 80 kW36,37. The thickness of the coatings was measured by a Vernier caliper to be about 0.10 mm.

Figure 1. The 10% Sr-nHA powder (A) and its plasma sprayed coating (B, right) on titanium rod (B, left). In (B) the scale bar = 2 mm.

Microstructure characterization Phase analyses of the Sr-nHA powders were characterized by X-ray diffraction (XRD, CuKα, 40 kV, 40 mA, Rigaku D/max-2550). The Fourier transform infrared (FTIR) spectra were obtained at 2 cm-1 intervals in the range of 800-4000 cm-1, using a spectrophotometer (Avatar 380, USA). The morphology of Sr-nHA powders was


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observed by transmission electron microscopy (TEM, Hitachi, H-7650). In addition, scanning electron microscopy (SEM, TM-1000, Hitachi) with energy dispersive of X-ray analysis (EDX) and XRD were used for surface microstructure observation and phase analysis of the Sr-nHA coatings, respectively. In addition, we soaked the coated samples in 15 mL Dulbecco’s Modified Eagle Medium (EMEM, Invitrogen, California) culture at room temperature for 0, 7 and 14 days (3 coated titanium rods for one group, length 15 mm, without refreshing the medium), and the concentrations of calcium and strontium ions were measured by ICP-MS (NexIONTM 350D, PE). For each group, we repeated three times. Cytotoxicity tests Cell isolation and culture Bone marrow-derived mesenchymal stem cells (BMSCs) were isolated from rabbits and cultured in DMEM (Invitrogen, California), supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Scientific, Massachusetts) and 1% penicillin and streptomycin (Gibco, Thermo Scientific, Massachusetts)38. In addition, primary osteoblasts of calvaria of rabbits were isolated according to a published protocol39,40. In addition, L-929 (murine fibroblast cells) and MC3T3-E1 (murine calvarial preosteoblasts) were also employed for the cytotoxicity study. The BMSCs of rabbits were passed three times, harvested and then plated for experiments. The primary osteoblasts in the 5-6 passage were seeded on 96-well plates at a density of 4000 cells per well and cultured normally for 24 h to facilitate cell attachment. BMSCs, MC3T3-E1 and L-929 were seeded at 3000 cells per well, as they proliferated much faster than the osteoblasts. Extract preparation Specimens (titanium rods with coatings) were immersed into DMEM or α-MEM (Invitrogen, California) supplemented with 10% FBS (Gibco, Thermo Scientific, Massachusetts) for 72 h under cell culture conditions (5% CO2, 95% humidity, 37 °C) with a fixed mass ratio to medium volume (0.2 g/ml) for preparing extracts according to ISO 10993 Part 1241. Cell proliferation assay Cell proliferation assays were performed by Cell Counting Kit-8 (CCK-8)42,43. Ten μl CCK-8 (Dojindo Molecular Technologies, Inc) was added to each well and incubated at 37 °C for 2 h. The absorbance was detected at a wavelength of 450 nm



by a plate reader. The primary osteoblasts were incubated for 1, 3 and 5 days under cell culture conditions, whereas viability tests for the other three cell types were performed on the first and third day. For cytotoxicity tests, experiments were repeated 3 times, and each group at each time point was replicated (n=5) to evaluate the toxic responses of the cells. Histocompatibility evaluation All animal experiment protocols conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication Eighth Edition, updated 2011). Male Sprague Darley rats (provided by Center of Experiment Animal of Xi’an Jiaotong University Health Science Center, weight 200-300 g). Ethical approval was obtained for the animal tests, which were conducted according to the ISO 10993-2:1992 Animal Welfare Requirements. The coated titanium rods (length 10 mm, diameter 1.2 mm) after disinfection were implanted into hind leg muscles of rats parallel to the muscle texture. Twenty-four male rats were randomly and equally divided into two groups for one week and two weeks after surgery (each time point, n=12). At each time point, four subgroups corresponding to 0%, 5%, 10% and 20% Sr-nHA (n=3, respectively) were divided equally and randomly. Twelve rats were sacrificed at one and two weeks after surgery, respectively. Three samples (rod and its surrounding muscles) from each subgroup were fixed in 4% formaldehyde and embedded in methyl methacrylate (Technovit 7200, Kulzer, Germany); then, slices of 3 μm in thickness were prepared vertically to the rod by using a cutting-grinding system (Exakt, Norderstedt, Germany). The sections were stained by HE staining for analysis of the associated tissue response to the implant. General toxicity test The coated titanium rods (length 20 mm, diameter 1.2 mm) were implanted into the distal femur of the rats according to the animal study procedure (Figure S1). Twenty-four male rats were randomly and equally divided into two groups for one week and two weeks after surgery (each time point, n=12). At each time point, four subgroups corresponding to 0%, 5%, 10% and 20% Sr-nHA (n=3, respectively) were divided equally and randomly. Twelve rats were sacrificed at one and two weeks after surgery. Heart, lung, liver, brain, kidneys and spleen were fixed in 4% formaldehyde and then embedded in methyl methacrylate (Technovit 7200, Kulzer, Germany); then, slices of 3 μm in thickness were prepared along the direct axis by using a


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cutting-grinding system (Exakt, Norderstedt, Germany). Hematoxylin and eosin (HE) staining was used for analyzing the associated tissue response to the implant. Mineralization test in vitro Titanium rods coated with Sr-nHAs were immersed in simulated body fluid (SBF) after being cut into the 20-mm-long rods. Each proportion of Sr-nHA sample was assigned on the third and tenth day for weighing, observing variations, and examination with SEM and XRD. Each proportion included twenty-four samples, evenly and randomly assigned into two subgroups (the third and tenth day group, n=12 respectively). In each subgroup, 6 of the twelve samples were used for checking weight variation. The remaining 6 samples were randomly assigned into two subgroups. One was for SEM, and the other one was for XRD. SBF was changed every day. On the third day, samples in each group were removed from the solution and ultrasonically washed with distilled water, dried under flowing air, and weighed by an analytical balance; then, the samples in each group were subjected to SEM and XRD analyses. On the tenth day, the samples were subjected to the same treatment as the third day of immersion in the SBF. Promotion of osteogenesis by Sr-nHA coating in vitro ALP activity assay The hBMSCs (Human bone marrow mesenchymal stem cells, purchased from Shanghai Cell Bank of Chinese Academy of Science) were cultured with 0%, 5%, 10% and 20% Sr-nHA coatings, respectively (length of the rod was slightly longer than the diameter of the well) in 24-well plate (5×104 cells per well). In addition, an osteogenic induction supplement group (OS group) that hBMSCs cultured with osteogenic induction supplement containing 5.0 mM β-glycerophosphate, 50 μg/mL ascorbic acid and 0.1 μM dexamethasone (Sigma-Aldrich, Missouri). The medium was refreshed every two days. Cells were harvested after 7, 10, 14 days and lysed by three cycles of freezing and thawing for ALP activity assay. The supernatants were subjected to ALP activity and total protein quantification measurement by an ALP activity kit (Dojindo Molecular Technologies, Tokyo). Western blotting The hBMSCs were cultured with 0%, 5%, 10% and 20% Sr-nHA coatings for 7 days. Then cells were lysed by the protein extraction buffer, and the supernatants were mixed with loading buffer containing SDS, dithiothreitol and bromophenol blue. 10% SDS-PAGE were used to separate protein after boiled for 10 min. The protein



was transferred to a PDVF membrane (Invitrogen, California). Then PDVF was blocked in 5% (w/v) non-fat powdered milk in TBST for one hour at room temperature and probed with diluted primary antibody at 4 °C overnight. The membrane was washed for three times. The membrane was incubated with secondary antibody for 1 h at room temperature. The enhanced chemiluminescent detection system was used for detecting protein levels. A CCD camera was employed to take the photos. The antibody was listed as follows: anti-runt related transcription factor 2 (RUNX2) antibody (ab135674), anti-bone morphogenetic protein 2 (BMP2) antibody (ab14933), anti-osteomodulin (OMD) antibody (ab16939), anti-CD44 antibody (ab157107), anti-endoglin (ENG) antibody (ab127607) and anti-GAPDH antibody (ab8245) were purchased from Abcam Biotechnology UK. Quantitative PCR (Q-PCR) The hBMSCs were cultured in 6-well plate (1×105 cells per well) and grown overnight, incubated with 20% Sr-nHA coating (length of the rod slightly longer than the diameter of well) for 7 days, the medium was refreshed every two days. Trizol (Sigma-Aldrich, Missouri) was used for isolating total RNA from cells according to the manufacturer’s protocols. Q-PCR was processed with following procedure: a denaturation step at 95 °C for 10 min followed by 40 cycles of 13 s at 95 °C and 60 s at 60 °C. The relative quantification of gene expression was determined by 2-△△T method. All reactions were in triplicate and performed in 20 reaction volume containing cDNA, primers and SYBR-GREEN mix (Takara Bio Compamy, Tokyo). The information of gene-specific primers was shown in Table S1. Animal study Animal model 156 male rats were conventionally raised for a week and randomly assigned into four groups (0%, 5%, 10% and 20% Sr-nHA, each group n=39). Ketamine (40-90 mg/Kg) and xylazine (5-10 mg/Kg) were used for animal anesthesia by intraperitoneal injection. After anesthesia came into effect, the hair on the knee joint was shaven, and the area was disinfected (Figure S1 A). Through a left medial parapatellar approach, 1.0 cm of skin incision was made, and subcutaneous tissue was dissected. The joint capsule was opened, and the patella was retracted to the lateral side; then the knee joint flexed to expose the femoral condyle (Figure S1 B). A drill with diameter of 1.0 mm was used to drill along the direction of the cavity of the femoral shaft slowly and


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uniformly, and once the resistance suddenly dropped, the drilling continued for 1.5 cm (Figure S1 C). The channel was slightly enlarged by a mold rod (diameter 1.2 mm). Finally, the titanium rod was tightly inserted without large friction (Figure S1 D). The patella was returned to its original placement, and the opening was sutured layer by layer. Penicillin of 20,000 units was injected intramuscularly after the operation. Twelve rats of thirty-nine in each group were sacrificed soon after surgery for biomechanical tests. The remaining ones were sacrificed five weeks after surgery. In each group, three were sacrificed for histological observation, six for the torsion-out test, six for the pulling-out test, six for the micro-CT, three for fluorescence microscopy observation and three for EDX analysis. Observations with van Gieson staining General observation Samples (n=3) in each group underwent van Gieson (V-G) staining for histological observation to evaluate bone formation in two regions (femoral distal cortex region and femoral distal region of epiphysis) after five weeks of surgery. In brief, the samples were stained in commercial 3.5% formalin for 5 days at room temperature, and then, the sample was dehydrated with gradient alcohol over 2 h; finally, the samples were embedded for V-G staining. The section slices, which included the bone cavity, were made through the titanium rod with a thickness of approximately 0.5 mm from the femoral distal region of epiphysis to the femoral distal cortex through the titanium rod44. Quantified observation Image Pro Plus 6.0 (Media Cybernetics, USA) was used to quantify the new bone formation in a comparison of the four groups. First, we randomly chose six histology slices from each group (0%, 5%, 10% and 20% Sr-nHA) five weeks after surgery. Second, we obtained the images in TIF format, and we exported them into Image Pro Plus 6.0 (Media Cybernetics, USA). Then, we divided the bone tissue area using Image Pro Plus 6.0. Finally, we calculated the area of separated bone tissue. Each sample was measured for three times. Fluorescence microscopy Fluorescent labeling was performed with tetracycline hydrochloride (SigmaAldrich, Missouri) protected from light. The tetracycline hydrochloride was subcutaneously injected into the neck of rats at a dose of 25 mg/kg. All animals were injected subcutaneously with tetracycline hydrochloride, as the first fluorescent



marker, on the 14th and 13th day before the end of the experiment. It was injected once more subcutaneously (25 mg/kg) on the fourth and third day before the end of the experiment as a second fluorescent marker, and a yellow double fluorescent mark was formed on the surface of the bone. The interval between the two fluorescent markers was 10 days, and bone formation during injections was dynamically observed. Under the fluorescence microscope, the tetracycline deposited on the surface of the bone was yellow. Biomechanical test Samples of femurs with titanium rods (n=24) in each group used for biomechanical tests were thawed at 4C for one night prior to testing. All surrounding soft tissues on the knee joints were removed carefully, and then, only the titanium rod and the femur remained. Pulling-out and torsion-out tests were conducted on an electronic universal material testing machine (Sanss-503, MTS, USA) soon after surgery and five weeks after surgery. All samples were fixed with two special designed clamps on the electronic universal material testing machine, and the assembly was loaded with a tensile speed of 5 mm/min and a force range of 10-250 N. Load-to-failure tests were used to determine the pulling-out force (PF). Torsion-to-failure tests were used to determine the torsion-out force (TF). During the testing, all specimens were moistened with normal saline. The environmental temperature was 26 C, and the relative humidity was 65%45. Micro-CT scanning After the muscular tissues attached above the femur were carefully removed, these bone samples were wrapped with gauze soaked in sodium chloride. The samples were stored at 20 C before scanning. All specimens (n=6) in each group were scanned with a high-resolution micro-CT tomography system (Y.Cheetah, Yxlon, Germany) in multislice and standard resolution mode46. A bone sample was placed horizontally in the sample chamber. In the beginning, the undesirable effect of the metal (titanium) on the image was removed by anti-artifact technology. Distal femurs were scanned along their long axes to obtain the images with 9.5 mm pixel size, and 180° tomography rotation scanning was selected to ensure image quality47. All scans were performed at voltage of 70 kV, current of 139 mA, aluminum filter of 1 mm, and rotation step of 1.6. All scans were reconstructed with the same parameters. The region of interest (ROI) was defined as the space of cavity around the titanium rod


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with a 3 mm height in the longitudinal section (Figure S2). Then, we calculated the trabecular microstructural parameters of the ROI, including bone tissue mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp)48. Interface observation by micro-CT and EDX The integrated interface between Sr-nHA coated titanium rod and bone was investigated by micro-CT and EDX. For micro-CT observation, we observed the area that included 300 μm around the titanium rod surface and a height of 2 mm like a cylinder. Longitudinal sections of samples (n=3) of each group were stained in SEM stationary liquid at 4 °C for a week. Then, all samples were dehydrated with deionized water, dried with a freeze-dryer, sprayed with gold, and then observed by EDX. 45 Statistical analysis All quantitative data were expressed as the mean ± SD. For comparison of data distributed in normal distribution between two groups, unpaired Student's two-tailed t-test was performed. Data distribution was tested previously by Shapiro-Wilk test. For analysis of comparison between multiple groups, one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test was performed. All the analysis was performed by using GraphPad Prism version 6.01 for Windows, GraphPad Software, La Jolla, California, USA. For all comparison analysis, differences were considered as significantly different for P < 0.05.

Results Microstructure characterization The typical absorption bands of the Sr-nHA powders were observed by FITR (Figure 2A). These results showed that no change occurred in the chemical structure of Sr-nHA after strontium addition. Weak bands of CO32− and OH- were also observed. The intensity of absorption peaks decreased with the increase of strontium content. Compared with the ICDD file of #9-0432, XRD peaks of HA were clearly identified for the synthesized Sr-nHA powders (Figure 2B). The grain size was 17.2, 16.8, 16.6 and 15.4 nm calculated from (002) peak for 0%, 5%, 10% and 20% Sr-nHA powders, respectively. The degree of crystallinity also decreased with the increase of strontium content according to the full width at half maximum (FWHM)



of (002) peak of HA. The TEM images showed that the synthesized nanoparticles had a tiny rod-like feature (Figure 2C). The tiny nanorods were 32.7 ± 8.8 nm, 34.2 ± 11.4 nm, 37.3 ± 12.7 nm, 40.5 ± 12.3 nm in length and 12.5 ± 2.7 nm, 14.0 ± 4.3 nm, 15.7 ± 4.7 nm, 16.0 ± 2.6 nm in width for 0%, 5%, 10% and 20% Sr-nHA powders, respectively (Figure 2C.a to Figure 2C.d), which were obtained by calculation with Image-Pro Plus version 6.0 software. The crystal sizes of each group had significant difference (P < 0.05) (Figure 2D).

Figure 2. A shows the absorption bands of the Sr-nHA powders. Weak bands of CO32− and OHwere also observed. The intensity of the absorption peaks decreased with the increase of Sr content. B shows that XRD patterns of Sr-nHA powder samples. C shows the TEM images of the Sr-nHA powders, in which the nanoparticles had a tiny rod-like feature (a-d, 0%-20% Sr-nHA, respectively). D shows the length and width values of tiny nanorods of the powder samples. Significant differences were observed among them. * means P < 0.05.

The XRD patterns of the coatings revealed that the main components were similar to those of the powders (Figure 3A). Significant deviation after spraying was not observed. The diffraction peaks could be clearly indexed for all the coating samples. The coating surface was rough, without any cracks or gaps (Figure 3B a,c,e,g). With the increase of strontium content, the coatings became rougher. Graphs of higher magnification showed that many uneven granules and deposits with irregular shape were located on the granules (Figure 3B b.d.f.h). Many micropores were seen among


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the granules and the irregular depositions. It was found that as the content of strontium increased, the micropores, granules and deposits with irregular shape increased. Elemental composition of the coatings was analyzed by EDX. It was clearly showed that the coatings contained the main elements of Ca, Sr, O and P (Figure 3C). The atomic ratio of (Ca + Sr)/P was approximately 1.67, which was the Ca/P atomic ratio of stoichiometric HA. The atomic ratios of Sr/(Ca + Sr) were approximately 5%, 10% and 20%, respectively for the Sr-nHA coatings (Table S2), which were in agreement with the initial design. In addition, the concentrations of Sr and Ca ions revealed an increasing trend from 0% to 20% Sr-nHA samples (Figure S3). In the first week, the releasing speeds of the four groups were faster than those in the second week, respectively (Figure S3).

Figure 3. A shows the XRD patterns for the coatings. No significant deviations were observed after spraying. B shows SEM images for the 0% (B. a,b), 5% (B.c,d), 10% (B.e,f) and 20% (B.g,h) Sr-nHA coatings. General observation (B.a, B.c, B.e, B.g) showed rough surface of the coatings without any cracks or gaps. Graphs of higher magnification (B.b, B.d, B.f, and B.h) showed uneven granules and deposits of irregular shapes located on the granules. Many microporous structures were seen among the spheres and irregular deposits. Red arrow: uneven granules. Yellow arrow: deposits of irregular shapes. C shows the EDX results of the coatings. The coatings contained the main elements of Ca, Sr, O and P. C.a to C.d correspond to the 0% to 20% Sr-nHA coatings. Scale bar of low magnification was 1 mm and that of high magnification was 100 μm.

Cytocompatibility tests



For the four types of cells, the extracts prepared by soaking the four kinds of coated samples in the cell culture medium showed no toxic effect, and the average viability of the four cell types at each time point was greater than 75% (Figure 4). No significant difference (P > 0.05) was revealed among the subgroups. According to Part 5 of the current ISO standards, cell viability greater than 75% could be considered with no toxic risks for medical devices.

Figure 4. Results of the CCK8 test. A-D correspond to the primary osteoblasts, BMSCs, MC3T3-E1 and L-929, respectively. For the four cell types, the extracts prepared from the coated titanium rods showed no toxic effect, and the average viability of the four cell types at each time point was greater than 75%. No significant difference was observed among the groups (P > 0.05).

Histocompatibility evaluation Hematoxylin and eosin-stained slices showed no inflammatory cell infiltration or necrotic tissue (Figure 5) one and two weeks after surgery. The Sr-nHA coatings on the titanium rod showed good histocompatibility.


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Figure 5. HE staining slices showed no inflammatory cell infiltration or necrotic tissue one and two weeks after surgery. Sr-nHA coatings on the titanium rod showed good histocompatibility. Red triangle is the position of implant. Scale bar = 100 μm.

General toxicity tests One week and two weeks after surgery, HE staining of the heart, lung, liver, brain and kidney of the rats showed that neither inflammatory cell infiltration nor necrosis of the tissue was observed, and microstructure of the organs was normal and easily identified (Figure 6). The coated titanium rods did not reveal a general risk of toxicity.

Figure 6. HE staining of the heart, lung, liver, brain and kidney organs of the rats for the 0%, 5%, 10% and 20% Sr-nHA groups one week and two weeks after surgery. Neither inflammatory cell infiltration nor necrosis of the tissue was observed, and microstructure of the organs was normal and easily identified. The Sr-nHA coatings did not reveal general risk of toxicity. Scale bar = 100



μm.

Mineralization experiment in vitro To show the weight variations more obviously, we combined five coated rods, like a bamboo pole (Figure 7A.a). After three days of soaking, the weight was 0.3560 ± 0.0520 g, 0.3650 ± 0.0400 g, 0.3530 ± 0.0470 g and 0.3558 ± 0.3675 g for 5% to 20% Sr-nHA samples, respectively. Ten days after immersion, the weight was increased to 0.3620 ± 0.0030 g, 0.3840 ± 0.0040 g, 0.3760 ± 0.0060 and 0.4120 ± 0.0190 g, respectively. From the line chart of Figure 7A.b, we could observe that the mineralization mass increased with the immersion time and also with the strontium content. The coating surface was rough, without any cracks or gaps. A large amount of white precipitate on the coating surface was observed (Figure 7B). With the increase of strontium content, the surface became rougher. The same trend was observed with the increase of mineralization time. From the in vitro mineralization test, we found 20% Sr-nHA sample was the best for apatite deposition. XRD analysis of this sample after ten days of immersion revealed similar diffraction pattern to that of nHA (Figure 7C).

Figure 7. Results of the mineralization test in vitro. A.a, where we combined five rods, like a bamboo pole. In A.b, we observe a trend showing the increase of mineralization mass with the immersion time and the strontium content. B shows the SEM images of the coatings after mineralization. The coating surface was rough, without any cracks or gaps. A large amount of white precipitate on the coating surface was observed. The surface became rougher with the increase of strontium content and immersion time. Scale bar of low magnification was 1 mm and that of high magnification was 100 μm. C is XRD pattern of 20% Sr-nHA sample after 10 days of immersion, which is similar to that of nHA.


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Promotion of osteogenesis by Sr-nHA coating in vitro treatment The activity of ALP revealed a dose-dependent effect from 0% to 20% Sr-nHA coatings and a time-dependent effect from 7 days to 14 days (Figure 8A). 20% Sr-nHA group showed obvious promotion compared with 0%, 5% and 10% Sr-nHA groups. Compared with 0% Sr-nHA group, 5% and 10% Sr-nHA groups revealed significant difference. The activity of ALP of 20% group was nearly the same as the OS group on the 7th and 14th day. The expression levels of osteogenic protein markers including RUNX2, BMP2 and OMD were up-regulated after the treatment with Sr-nHA coatings in vitro and showed a dose-dependent effect from 0% to 20% Sr-nHA coatings (Figure 8B), while the expression levels of hBMSCs protein markers including CD44 and ENG were down-regulated from the 0% to 20% Sr-nHA coatings (Figure 8B). Compared with 0%, 5% and 10% Sr-nHA coatings, 20% Sr-nHA group showed obviously higher RUNX2, BMP2 and OMD and significantly lower ENG and CD44 (Figure 8C, 8D). The expression levels of RUNX2 and BMP2 determined by immunofluorescence in vivo showed the same trend with those in vitro (Figure S4, Figure S5). For 20% Sr-nHA coating, the expression levels of mRNA including BMP2, RUNN2 and OMD were up-regulated by 7-15 folds (Figure 8E), while those of CD44 and ENG was down-regulated nearly by 0.5 folds (Figure 8F).



Figure 8. The results of promotion of osteogenesis by Sr-nHA coatings in vitro. A shows the effect of Sr-nHA coatings on the ALP of hBMSCs. B is the expression of protein level detected by western blotting after 7 days of Sr-nHA coating treatment. C and D are the quantified results of western blotting. E and F are expression level of mRNA detected by Q-PCR after 20% Sr-nHA


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coating treatment for 7 days. All bars represent mean ± SD, n=3. *p < 0.05; **p < 0.01; ***P < 0.001.

V-G histological observation Figure 9.1 shows that V-G of the femur with 0% Sr-nHA rod revealed relatively slow bone formation around the rod five weeks after surgery near the region of the epiphysis (Figure 9.1A, B were from 0% Sr-nHA). More new red mature bone was observed with V-G in the femur with 20% Sr-nHA (Figure 9.1M, N) than that in the graph of the 0% (Figure 9.1A, B), 5% (Figure 9.1E, F) and 10% (Figure 9.1I, J) Sr-nHA group. The cortex region with V-G staining showed the same trend as the region of epiphysis. V-G staining of the femur with 0% Sr-nHA (Figure 9.1C, D) revealed a relatively slow bone formation around the rod five weeks after surgery in the cortex area. More new mature bone was observed with V-G staining of the femur with 20% Sr-nHA (Figure 9.1O, P) than that in the graph of 0% (Figure 9.1C, D), 5% (Figure 9.1G, H), 10% Sr-nHA group (Figure 9.1K, L).

Figure 9.1. V-G staining of the femur five weeks after surgery. A, E, I and M show the epiphysis region of the 0%, 5%, 10% and 20% groups, respectively, five weeks after surgery. C, G, K and O show the cortex region of the 0%, 5%, 10% and 20% group, respectively, five weeks after surgery. B, F, J, N show the zoom view of A, E. I, M. D, H, L, P are the zoom view of C, G, K, O. Red-stained tissues (red arrow) are mature bone tissues. Purple-stained tissues (green arrow) are new immature bone tissues. The black rod (yellow arrow) is the implant. Purple arrow: lineae epiphysialis. Black arrow: cortex bone. Scale bar of 16X and 50X are about 500 μm and 200 μm.



For example, we chose Figure 9.2B to calculate the mature bone area. In Figure 9.2C, the rod was deleted from the picture. Figure 9.2D shows the mature bone area only. Figure 9.2A shows the results of the quantified V-G staining. The average quantified bone area sizes of the epiphysis of the 20% to 0% Sr-nHA groups were 205284 ± 58214 units, 186366 ± 46720 units, 167379 ± 66940 units and 127366 ± 55920 units, respectively. The average quantified bone area sizes of cortical region of the 20% to 0% Sr-nHA groups were 124306 ± 35558 units, 104101 ± 25528 units, 69670 ± 14797 units and 49770 ± 24797 units, respectively. 20% Sr-nHA group showed the highest area compared with 0%, 5% and 10% Sr-nHA. Compared with 0% Sr-nHA group, 5% and 10% Sr-nHA groups revealed a significant difference (Figure 9.2 A).

Figure 9.2. A is the results of quantified histological observation. CR: Cortical region. ER: Epiphyseal region. B, C and D are the procedures of calculating area of one sample. A is one slice of 10% Sr-nHA. B shows the titanium rod was deleted from the picture. D shows the mature bone area only. All bars represent means ± SD, n=6. * means P < 0.05. ** means P < 0.01. *** means P < 0.001.

Fluorescence microscopy observation Five weeks after surgery, the two golden-yellow lines, which were approximately parallel, were observed by fluorescence microscopy (Figure 10). These observations showed the labeling was successful and the bone formation was a dynamic process. We observed deviations in the gap of the two lines in the 0%, 5%, 10% and 20%


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Sr-nHA groups in the cortical region. The mineral apposition rate (MAR) calculated by MAR=D/T, with two-line interval width (D) and time interval (T). The MAR value was 22.6 ± 2.1 μm·d-1, 25.8 ± 3.2 μm·d-1, 26.4 ± 2.3 μm·d-1 and 35.5 ± 1.3 μm·d-1 for 0%, 5%, 10% and 20% Sr-nHA groups, respectively. 20% Sr-nHA group showed the highest MAR than those of 0%, 5% and 10% Sr-nHA groups. Compared with 0% Sr-nHA group, 5% and 10% Sr-nHA groups revealed significant difference (Figure 10B).

Figure 10. Fluorescent microscopy observation for bone forming. Two yellow lines (red arrow) indicate depositions of tetracycline. A is the measurement of the gap between two golden lines, and B is a comparison of the results. * means P < 0.05. ** means P < 0.01. *** means P < 0.001. Scale bar of 200X is 100 μm. Scale bar of 400X was 200 μm.

Micro-CT scanning



The rods were in the cavity without any motion (Figure S6 A-C). It was easy to select ROI for calculation without artifacts. The BMD of the ROI (Figure S6 D, E, F, G) was 418.6 ± 24.1 mg/cc, 470.3 ± 19.4 mg/cc, 480.4 ± 21.6 mg/cc, 546.0 ± 27.6 mg/cc for 0%, 5%, 10% and 20% groups, respectively. 20% Sr-nHA group showed the highest BMD value compared with 0%, 5% and 10% Sr-nHA groups (Figure 11A). Compared with 0% Sr-nHA group, 5% and 10% Sr-nHA groups revealed a significant difference. The other parameters like BV/TV, Tb.Sp, Tb.n and Tb.Th were shown in Figure 11 B, C, D, E. Interface observation Figure 12 revealed that the new trabecular appeared around the titanium rod five weeks after surgery. From 0% to 20% Sr-nHA groups, the number of trabecular showed an increasing trend and the 20% Sr-nHA group might be the best. The areal and linear scanning results at the longitudinal section (Figure S7, Figure 13 and Figure 14) indicated that calcium and phosphorus elements appeared around the coated titanium rods. The concentration of calcium and phosphorus around the titanium rods revealed an increasing trend from 0% to 20% Sr-nHA groups. The strontium element also appeared around the titanium rod and it showed the same distribution as calcium and phosphorus elements.

Figure 11. Results of the micro-CT observation. A is BMD of ROI. B is BV/TV of ROI. C is Tb.Sp of ROI. D is Tb.N of ROI. E is Tb.Th of ROI. * means P < 0.05. ** means P < 0.01. *** means P < 0.001.


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Figure 12. Three-dimensional reconstruction of new trabecular appeared around the titanium rods (300 μm) five weeks after surgery. From 0% to 20% Sr-nHA groups, the number of the trabecular showed an increasing trend. A, C, E, G are the bone with coated titanium from 0% to 20% Sr-nHA groups. B, D, F, H are the bone tissue only from 0%-20% Sr-nHA groups. Scale bar=300 μm.

Figure 13. The areal EDX scanning at longitudinal section. The density of Ca and P around the titanium rod revealed an increasing trend from 0% to 20% Sr-nHA group. The strontium element also appeared around the titanium rod and showed the same change as the calcium and phosphorus elements. Scale bar=900 μm.



Figure 14. The linear EDX scanning along the width direction at longitudinal section (Figure S7). Calcium, phosphorus and strontium elements appeared around the titanium rods. The density of calcium and phosphorus around the titanium rods revealed an increasing trend from 0% to 20% Sr-nHA groups. The strontium element also appeared around the titanium rods and showed the same distribution as calcium and phosphorus elements.

Biomechanical test The pulling-out force was 52.0 ± 9.8 N, 49.0 ± 7.1 N, 50.0 ± 8.4 N, 48.0 ± 8.8 N soon after surgery and 123.0 ± 11.9 N, 189.2 ± 21.1 N, 212.4 ± 16.5 N, 241.8 ± 11.4 N five weeks after surgery for 0%, 5%, 10% and 20% Sr-nHA groups, respectively (Figure 15A). However, pulling-out force soon after surgery showed no significant difference among the groups. The pulling-out force showed a significant difference between the two time points. Five weeks after surgery, 5% and 10% Sr-nHA groups revealed a significant difference compared with 0% Sr-nHA group, and 20% Sr-nHA group showed the highest pulling-out force among the four groups. It suggested that 20 at.% Sr might be the suitable dosage in decreasing aseptic loosening of femoral prosthesis in vivo. The torsion-out force was 51.5 ± 6.0 N, 52.6 ± 6.3 N, 49.6 ± 6.1 N, 48.7 ± 6.5 soon after surgery and 145.2 ± 12.2 N, 168.3 ± 10.7 N, 178.7 ± 11.4 N, 241.8 ± 11.4 N five weeks after surgery for the 0%, 5%, 10% and 20% Sr-nHA groups, respectively (Figure 15B). The torsion-out force showed the same trend with the pulling-out force.


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Figure 15. Results of biomechanical test. A is the pulling-out force of the four groups soon after surgery and five weeks after surgery. B is the torsion-out force of the four groups soon after surgery and five weeks after surgery. All the bars represent mean ± SD, n=6. *** means P < 0.0001.

Discussion In this study, we focused on the utilization of plasma sprayed strontium-substituted nanohydroxyapatite coatings on titanium rod to solve a tough problem: aseptic loosening of the femoral prosthetic stem after hip replacement. The Sr-nHAs revealed no toxic risk when used as coating material. Employing Sr-nHA coating on titanium implants for surface modification showed safety and great application potential in decreasing the aseptic loosening of femoral prostheses after hip replacement. This coating provides a method for decreasing aseptic loosening of femoral prostheses. For material characterization, the weak absorption bands of CO32− in FTIR spectra indicate the existence of CO32− groups, which came from the air during preparation of the powders. The incorporation of CO32− groups is often observed in HA preparation49, making the composition similar to natural apatite. When strontium was added to the nanohydroxyapatite, the crystal structure changed substantially. First, we found that the grain size of Sr-nHA showed an decreasing trend with the increase of strontium content. This was consistent with the results of previous studies50,51. The degree of crystallinity showed the same decreasing trend, which was also consistent with the report by Ni et al.52. The prepared Sr-nHA powders remained their nanograined structure after being coated on titanium rods. We employed the SBF test to observe mineralization process of the Sr-nHA coated samples in vitro. From SEM observation, we found that the surface became rougher with the increase of strontium content and immersion time. The sample mass increment and XRD analysis demonstrated the deposition of apatite layer on the sample surface, especially for the coated samples with high Sr content. The increasing ALP activity is an indispensable marker in early stage of osteogenesis (about 7-14 days)53-55. It has been demonstrated that several markers



were inevitably abnormally expressed during the course of osteogenesis55. The activity of ALP in this study increased significantly with strontium content and culture time from 4 to 14 days. In western blotting, the osteogenic protein markers were also up-regulated, but the hBMSCs’ protein markers were down-regulated from 0% to 20% Sr-nHA groups. Furthermore, the mRNA expression levels of these osteogenic markers was much higher, while the mRNA expression levels of hBMSCs’ markers was much lower than that of the control group. Therefore, the mineralization and the cell tests in vitro demonstrated the safety and efficacy of Sr-nHA coatings. Our cytology experiment results were consistent with the studies performed by Zhou et al. published in ACS Applied Materials & Interfaces56 and Chung et al. published in Acta Biomaterialia57. Zhou et al.56 explored the quantity of protein and its corresponding mRNA of osteoblasts grown on Sr-HA coating. Two kinds of Sr-HA coating samples with different surface topography (nanoparticles and nanobars) on titanium substrate were prepared and cultured with human embryonic osteoblast as the samples. Both the quantity of the investigated osteogenic proteins and the quantity of corresponding mRNA increased with the prolongation of culture time, and the increments are more obvious for the nanorod-like coating. Chung et al.57 prepared a series of Sr-HA coatings on titanium sheet by electrochemical deposition, and cultured RAW 264.7 osteoclasts on Sr-HA coatings. When the ratio of Sr/(Ca+Sr) exceeded 38.9%, the Sr-HA coatings appeared to show inhibition of the osteoclasts, which agreed with the publication in Rheumatology by Fonseca et al.58 In animal experiments, the results of histological observation, fluorescence microscopy, microcomputed tomography, biomechanical test and scanning electron microscopy indicated that 0%, 5%, 10% and 20% Sr-nHA groups had good safety and efficacy. The 20% Sr-nHA group performed the best in bone formation and showed superior biomechanical properties to other groups. Our in vitro and in vivo experiment results strongly verified the safety and efficacy of Sr-nHA coatings that were used as a coating material for decreasing aseptic loosening. This study may provide a method for decreasing aseptic loosening of femoral prosthesis and lay the foundation for further large animal research.

Conclusions For the synthesized Sr-nHA powders, the grain size and the degree of crystallinity decreased with the increase of strontium content. The 0%, 5%, 10% and 20% Sr-nHA


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coatings on the titanium rods showed good biocompatibility, without any toxicity observed. In SBF test, the addition of strontium enhanced mineralization process of the coatings. The in vitro bone forming study and the animal study revealed safety and efficacy of the Sr-nHA coatings, especially for the 20% Sr-nHA coating. Strontium-substituted nanohydroxyapatite coatings on titanium showed great application potential for decreasing the aseptic loosening of femoral prostheses after hip replacement.

Limitations The current work demonstrated the benefits of Sr-nHA coatings. Since the release of strontium ions plays a crucial role in new bone formation process, it is worthy of studying the release kinetics of strontium ions released from the plasma sprayed Sr-nHA coatings. We just measured the concentrations of strontium and calcium ions at two time points. Additionally, the slow and continuous release of strontium ions would enable a stable and long-term effect to promote bone formation and inhibit bone resorption for the aspect of decreasing the aseptic loosening of femoral prostheses after hip replacement. How to keep strontium ions releasing stably and continuously was not explored in this study. We would prepare Sr-nHA coatings on micro-rough titanium to increase the quantity of the coating material to investigate the release kinetics of strontium ions in the following study.

Acknowledgments The authors wish to thank Prof. Zhang Dong for his expertise and kind help with SEM. Also we would like to thank Prof. Li Dichen for his kind help with the biomechanical test. We also thank Prof. Zhang Xuebin for his kind help with histological observation. We sincerely thank the National Key Research and Development Program of China (No. 2018YFC1106200/2/4), the National Natural Science Foundation of China (No. 81672187) and the Research Projects of Xi’an Jiaotong Universtiy (No. XJLS-2015-056, XJTU1AF-CRF-2016T-06) for funding our research and continuing to support us to go on this study.

Supporting Information Supporting Information Available: Procedures for establishing animal model; Primers information for Q-PCR; Definition of ROI; Releasing curve; EDX analysis results; Immunofluorescence of BMP2 and RUNX2; 3D reconstruction for new forming bone; Longitudinal section for SEM scanning.



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Table of content



TOC 160x130mm (300 x 300 DPI)


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Figure 1 The 10% Sr-nHA powder (A) and its plasma sprayed coating (B, right) on titanium rod (B, left). In (B) the scale bar = 2 mm. 80x39mm (300 x 300 DPI)



Figure 2 A shows the absorption bands of the Sr-nHA powders. Weak bands of CO32− and OH- were also observed. The intensity of the absorption peaks decreased with the increase of Sr content. B shows that XRD patterns of Sr-nHA powder samples. C shows the TEM images of the Sr-nHA powders, in which the nanoparticles had a tiny rod-like feature (a-d, 0%-20% Sr-nHA, respectively). D shows the length and width values of tiny nanorods of the powder samples. Significant differences were observed among them. * means P < 0.05. 179x150mm (300 x 300 DPI)


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Figure 3 A shows the XRD patterns for the coatings. No significant deviations were observed after spraying. B shows SEM images for the 0% (B. a,b), 5% (B.c,d), 10% (B.e,f) and 20% (B.g,h) Sr-nHA coatings. General observation (B.a, B.c, B.e, B.g) showed rough surface of the coatings without any cracks or gaps. Graphs of higher magnification (B.b, B.d, B.f, and B.h) showed uneven granules and deposits of irregular shapes located on the granules. Many microporous structures were seen among the spheres and irregular deposits. Red arrow: uneven granules. Yellow arrow: deposits of irregular shapes. C shows the EDX results of the coatings. The coatings contained the main elements of Ca, Sr, O and P. C.a to C.d correspond to the 0% to 20% Sr-nHA coatings. Scale bar of low magnification was 1 mm and that of high magnification was 100 μm. 160x136mm (300 x 300 DPI)



Figure 4 Results of the CCK8 test. A-D correspond to the primary osteoblasts, BMSCs, MC3T3-E1 and L-929, respectively. For the four cell types, the extracts prepared from the coated titanium rods showed no toxic effect, and the average viability of the four cell types at each time point was greater than 75%. No significant difference was observed among the groups (P > 0.05). 80x68mm (300 x 300 DPI)


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Figure 5 HE staining slices showed no inflammatory cell infiltration or necrotic tissue one and two weeks after surgery. Sr-nHA coatings on the titanium rod showed good histocompatibility. Red triangle is the position of implant. Scale bar = 100 μm.



Figure 6 HE staining of the heart, lung, liver, brain and kidney organs of the rats for the 0%, 5%, 10% and 20% Sr-nHA groups one week and two weeks after surgery. Neither inflammatory cell infiltration nor necrosis of the tissue was observed, and microstructure of the organs was normal and easily identified. The Sr-nHA coatings did not reveal general risk of toxicity. Scale bar = 100 μm.


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Figure 7 Results of the mineralization test in vitro. A.a, where we combined five rods, like a bamboo pole. In A.b, we observe a trend showing the increase of mineralization mass with the immersion time and the strontium content. B shows the SEM images of the coatings after mineralization. The coating surface was rough, without any cracks or gaps. A large amount of white precipitate on the coating surface was observed. The surface became rougher with the increase of strontium content and immersion time. Scale bar of low magnification was 1 mm and that of high magnification was 100 μm. C is XRD pattern of 20% Sr-nHA sample after 10 days of immersion, which is similar to that of nHA.



Figure 8 The results of promotion of osteogenesis by Sr-nHA coatings in vitro. A shows the effect of Sr-nHA coatings on the ALP of hBMSCs. B is the expression of protein level detected by western blotting after 7 days of Sr-nHA coating treatment. C and D are the quantified results of western blotting. E and F are expression level of mRNA detected by Q-PCR after 20% Sr-nHA coating treatment for 7 days. All bars represent mean ± SD, n=3. *p < 0.05; **p < 0.01; ***P < 0.001. 160x297mm (300 x 300 DPI)


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Figure 9.1 V-G staining of the femur five weeks after surgery. A, E, I and M show the epiphysis region of the 0%, 5%, 10% and 20% groups, respectively, five weeks after surgery. C, G, K and O show the cortex region of the 0%, 5%, 10% and 20% group, respectively, five weeks after surgery. B, F, J, N show the zoom view of A, E. I, M. D, H, L, P are the zoom view of C, G, K, O. Red-stained tissues (red arrow) are mature bone tissues. Purple-stained tissues (green arrow) are new immature bone tissues. The black rod (yellow arrow) is the implant. Purple arrow: lineae epiphysialis. Black arrow: cortex bone. Scale bar of 16X and 50X are about 500 μm and 200 μm.



Figure 9.2 A is the results of quantified histological observation. CR: Cortical region. ER: Epiphyseal region. B, C and D are the procedures of calculating area of one sample. A is one slice of 10% Sr-nHA. B shows the titanium rod was deleted from the picture. D shows the mature bone area only. All bars represent means ± SD, n=6. * means P < 0.05. ** means P < 0.01. *** means P < 0.001. 160x112mm (300 x 300 DPI)


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Figure 10 Fluorescent microscopy observation for bone forming. Two yellow lines (red arrow) indicate depositions of tetracycline. A is the measurement of the gap between two golden lines, and B is a comparison of the results. * means P < 0.05. ** means P < 0.01. *** means P < 0.001. Scale bar of 200X is 100 μm. Scale bar of 400X was 200 μm. 161x218mm (149 x 149 DPI)



Figure 11 Results of the micro-CT observation. A is BMD of ROI. B is BV/TV of ROI. C is Tb.Sp of ROI. D is Tb.N of ROI. E is Tb.Th of ROI. * means P < 0.05. ** means P < 0.01. *** means P < 0.001. 160x94mm (300 x 300 DPI)


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Figure 12 Three dimensional reconstruction of new trabecular appeared around the titanium rod (300 μm) five weeks after surgery. From 0% to 20% Sr-nHA groups, the number of the trabecular showed an increasing trend. A, C, E, G are the bone with coated titanium from 0% to 20% Sr-nHA groups. B, D, F, H are the bone tissue only from 0%-20% Sr-nHA groups. Scale bar=300 μm. 199x95mm (300 x 300 DPI)



Figure 13 The areal EDX scanning at longitudinal section. The density of Ca and P around the titanium rod revealed an increasing trend from 0% to 20% Sr-nHA group. The strontium element also appeared around the titanium rod and showed the same change as the calcium and phosphorus elements. Scale bar=900 μm. 160x139mm (300 x 300 DPI)


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Figure 14 The linear EDX scanning along the width direction at longitudinal section (Figure S7). Calcium, phosphorus and strontium elements appeared around the titanium rod. The density of calcium and phosphorus around the titanium rods revealed an increasing trend from 0% to 20% Sr-nHA groups The strontium element also appeared around the titanium rods and showed the same distribution as calcium and phosphorus elements.



Figure 15 Results of biomechanical test. A is the pull-out force of the four groups soon after surgery and five weeks after surgery. B is the torsion-out force of the four groups soon after surgery and five weeks after surgery. All the bars represent mean ± SD, n=6. *** means P < 0.0001. 160x48mm (300 x 300 DPI)


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Figure S1 The procedure of establishing animal model. A is the disinfection step. B shows the femoral condyle. C is drilling tunnel along the cavity. D shows that the titanium rod was slightly inserted in without robust friction.



Figure S2 The selection of the ROI defined as the space of cavity around the titanium rod with a 3 mm height in the longitudinal section. A is separating the cortical bone from the tissue around titanium rod in the cross-section. B is a selection of 3 mm height of the ROI (red line). 160x116mm (300 x 300 DPI)


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Figure S3 The releasing curve of Sr-nHA coatings soaked in the DMEM culture for 0, 7 and 14 days. A is the concentration of calcium ion. B is the concentration of strontium ion. Ca= calcium ion. Sr= strontium ion. All bars represent mean ± SD, n=3. 79x29mm (300 x 300 DPI)



Figure S4 The immunofluorescence of BMP2 in the tissue around the titanium rods five weeks after surgery. The brightness of the red fluorescent means the expression level of the BMP2 in tissue. Scale bar is about 200 μm. 160x90mm (300 x 300 DPI)


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Figure S5 The immunofluorescence of RUNX2 in the tissue around the titanium rods five weeks after surgery. The brightness of the red fluorescent means the expression level of the RUNX2 in tissue. Scale bar is about 200 μm. 224x127mm (149 x 149 DPI)



Figure S6 Image of micro-CT without any artifacts. A shows the sagittal view. B shows the coronal view. C shows the axial view. We note that, after the anti-artifact technique was applied, the image around the titanium metal was of high quality without any artifacts. Yellow arrow: titanium metal rod. D, E, F, G is the view of 3D reconstruction of ROI. 80x100mm (300 x 300 DPI)


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Figure S7 The longitudinal section of titanium rod and new forming tissue around it. A shows the linear scanning position. B is the areal scanning position. A and B were from the 20% Sr-nHA coating group.



Table S1 The gene-specific primers information 159x62mm (300 x 300 DPI)


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Table S2 The results of EDX analysis 161x64mm (300 x 300 DPI)


Strontium-substituted Nanohydroxyapatite Coatings to Decrease

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