Osteogenic ability of Cu-bearing stainless steel Ling Ren,1* Hoi Man Wong,2,3* Chun Hoi Yan,2,3 Kelvin W.K. Yeung,2,3 Ke Yang2 1

Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016, China Department of Orthopaedics and Traumatology, The University of Hong Kong, Pokfulam, Hong Kong, China 3 Shenzhen Key Laboratory for Innovative Technology in Orthopaedic Trauma, The University of Hong Kong Shenzhen Hospital, Shenzhen, China 2

Received 31 May 2014; revised 28 September 2014; accepted 18 October 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33318 Abstract: A newly developed copper-bearing stainless steel (Cu-SS) by directly immobilizing proper amount of Cu into a medical stainless steel (317L SS) during the metallurgical process could enable continuous release of trace amount of Cu21 ions, which play the key role to offer the multibiofunctions of the stainless steel, including the osteogenic ability in the present study. The results of in vitro experiments clearly demonstrated that Cu21 ions from Cu-SS could promote the osteogenic differentiation by stimulating the Alkaline phosphatase enzyme activity and the osteogenic gene expressions (Col1a1, Opn, and Runx2), and enhancing the adhesion and proliferation of osteoblasts cultured on its surface. The in vivo test further proved that more new bone tissue formed around the Cu-SS implant with more stable bone-to-implant contact in comparison with the 317L SS. In addition, Cu-SS showed satisfied bio-

compatibility according to the results of in vitro cytotoxicity and in vivo histocompatibility, and its daily released amount of Cu21 ions in physiological saline solution was at trace level of ppb order (1.4 ppb/cm2), which is rather safe to human health. Apart from these results, it was also found that Cu-SS could inhibit the happening of inflammation with lower TNF-a expression in the bone tissue post implantation compared with 317L SS. In addition to good biocompatibility, the overall findings demonstrated that the Cu-SS possessed obvious ability of promoting osteogenesis, indicating a unique application advantage in orthopedics. C 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl BioV

mater 00B: 000–000, 2014.

Key Words: stainless steel, metal ions, bone, osteogenesis, bioactive material

How to cite this article: Ren, L, Wong, HM, Yan, CH, Yeung, KWK, Yang, K 2014. Osteogenic ability of Cu-bearing stainless steel. J Biomed Mater Res Part B 2014:00B:000–000.

INTRODUCTION

Metallic biomaterials such as stainless steel, titanium alloy, and cobalt-based alloy are widely used in surgical implantations due to their good mechanical properties and corrosion resistance and sufficient biocompatibility. Indeed, over 70% of implant devices are made of metals, especially in orthopedics, for example, joint prostheses, spinal implants, bone fixation plates, and nails.1 However the osteogenic ability of metal implants is largely limited by the characteristic of their bioinertness.2 Thus, aseptic loosening and subsequent premature failure for the implants were documented.3–5 To overcome this clinical problem, researches have extensively developed various bioactive coatings on metal implants by adopting different surface modifications.6–9 A common strategy is to immobilize the bioactive elements such as calcium (Ca), silicon (Si), zinc (Zn), and strontium (Sr) into the surface coating through conventional coating techniques so that the bioactive elements can be released during implantation,

thereby improving the osteogenic ability of the metallic implants. For instance, Andersen et al.10 accelerated the bone ingrowth by local delivery of Sr ions from the surface functionalized titanium implants. Ballarre et al.11 incorporated Si in the sol–gel coating on stainless steel implants for improving the osteointegration. Despite the efforts made in this aspect, the delamination of coating on implant surface was still a concern, and a sustainable release of bioactive elements was also difficult to achieve.12,13 To address these technical difficulties, it seems that direct incorporation of bioactive elements into metal matrix during metallurgical process should be a more feasible solution instead of conventional surface coating technologies. Among various bioactive metal elements, Cu is not only an alloying element in many alloys such as steels, but also an essential trace element to maintain cellular functions in human body.14,15 Apart from the use as an antibacterial reagent in the previous studies,16,17 Cu21 ions are also

*Both authors contributed equally to this work Correspondence to: K. Yang; e-mail: [email protected] Contract grant sponsor: National Basic Research Program of China (973 Program); contract grant number: 2012CB619101 Contract grant sponsor: National Natural Science Foundation of China; contract grant number: 81301329 Contract grant sponsor: Shenyang National Lab for Materials Science

C 2014 WILEY PERIODICALS, INC. V

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beneficial to cardiovascular system by stimulating the proliferation of vascular endothelial cells (VECs), thereby improving the angiogenic process.18–23 Due to these attractive biofunctions of Cu element, a novel bio-functional metallic biomaterial, namely Cu-bearing stainless steel (Cu-SS), has been developed and was reported in previous studies.24–29 This novel metallic biomaterial is fabricated by immobilizing proper amount of Cu directly into the conventional medical stainless steels, such as 304, 316L, and 317L SS, during the steel making process. It was found that, a trace amount of Cu21 ions could release from the Cu-SS while exposing to the physiological environment,24,25 which will not affect its corrosion resistance according to the previous electrochemical corrosion measurement.24,25 Thus, the new stainless steel with Cu addition demonstrated strong antibacterial abilities in both orthopeadic26,27 and stomatological studies.28 For the cardiovascular application, it could even reduce the tendency of in-stent restenosis (ISR) due to the stimulation of VEC proliferation and the inhibition of vascular smooth muscle cells (VSMCs) and thrombus formation as well.29 Although the immobilization of Cu (4–5 wt %) into the stainless steels, such as 304, 316L, and 317L SS, offers their multiple bio-functions, the mechanical properties, uniform corrosion resistance, and microstructure of the Cu-SS are not significantly affected and almost the same as those of the relevant stainless steels.24,25 If this newly developed bio-functional Cu-SS with release Cu21 ions is considered for orthopedic implantation, its osteogenic ability is worth to be characterized, since some recent studies have shown that Cu21 ions could benefit to bone formation, which is great important for bone repairs. Therefore, the aim of this study was to investigate the osteogenic ability of this novel metallic biomaterial through a series of in vitro and in vivo experiments. MATERIALS AND METHODS

Materials A 317L-Cu stainless steel (Cu-SS), based on the chemical composition of currently used type 317L stainless steel (317L SS) in orthopedics, was designed and fabricated in our previous work.24–29 The Cu-SS, with nominal chemical compositions (wt %): Cr 19, Ni 13, Mo 3.5, Cu 4.5, and Fe in balance, was melted in a 25 kg capacity vacuum induction melting furnace. The cast ingot was reheated to 1100 C, held for 1 h, and then hot forged into plates. The forged plates were solution treated at 1040 C for 1h followed by a water quench, and then were aged at 700 C for 6 h. The samples for in vitro tests were machined into disks of 4.4 and 13.9 mm in diameters and 1 mm in thickness, respectively. Then, they were mechanically polished with silicon carbide papers down to 2000 grade and ultrasonically washed in acetone and ethanol. For the in vivo test, the samples were machined into cylinder shape of 2 mm in diameter and 6 mm in length followed by electrochemical polishing. All the experimental samples were sterilized before all the biological tests. Analysis of Cu21 ions released from Cu-SS To quantitatively determine the amount of Cu21 ions released from the Cu-SS in biological environment, the

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Cu-SS disc samples were immersed into physiological saline solution (0.9% NaCl solution with pH 5 7.4). The 10 samples containing in 3 mL of physiological saline solution were placed in a 37 C incubator for day 1, 6, 9, 22, and 30, respectively. Afterwards, an inductively coupled plasma atomic emission spectrometer (ICP-AES) (Thermo Elemental, IRIS Intrepid) was applied to quantify the amount of Cu21 ions released from Cu-SS samples in the collected physiological saline solutions. In vitro tests Cell morphology and morphometry. To examine the cellular activity of the new stainless steels, the enhanced green fluorescent protein osteoblasts (eGFPOB) from GFP mice were cultured on the samples surfaces, respectively. Each of disk samples with 4.4 mm in diameter was fixed on the bottom of 96-well tissue culture plate. A cell suspension consisting of 1.7 3 104 cells/cm2 eGFPOB was seeded directly onto the samples (n 5 3). Cells were cultured in the Dulbecco’s modified eagle medium (DMEM) (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (Biowest, France), antibiotics (100 U/mL of penicillin and 100 lg/mL of streptomycin), and 2 mM L-glutamine, which were incubated at 37 C in an atmosphere of 5% CO2 and 95% air. Cell attachment and proliferation were examined after 1 and 3 days of culture. Cells were allowed to reach a confluence during the examination period. Cell morphology was observed using a fluorescent microscope (Niko ECL IPSE 80i, Japan). The attached living eGFPOB-expressive osteoblasts were visualized using a 450–490 nm incident filter, and the fluorescence images were emitted at 510 nm and captured using a Sony DKS-ST5 digital camera. In addition, to analyzed the morphometry of the cells on the samples, an aliquot of 1.7 3 104 cells/cm2 SaOS-2 human osteoblasts was seeded on each sample and cultured for 1 day (n 5 3). After 1 day of culturing, the cells were washed with PBS, stained with calcein AM and incubated in an incubator for 10 min. The stained cells were also viewed on fluorescent microscopy after PBS washing and images were captured for analysis. The cell culture on 317L SS surface was served as control. The aspect ratio (the ratio of cell’s longer side to its shorter side) and area of attached cells were analyzed by Image J (version: 1.46). Cell viability assessment. The cytotoxicity of the new Cu-SS was determined by MTT assay. The samples of 13.9 mm in diameter were used (n 5 6). A total of 2 3 104 cells/cm2 SaOS-2 human osteoblasts were seeded on 317L SS and CuSS samples, respectively. The cells were cultured in the DMEM culture medium supplemented with 10% (v/v) fetal bovine serum, antibiotics (100 U/mL of penicillin and 100 lg/mL of streptomycin), and 2 mM L-glutamine and incubated at 37 C in an atmosphere of 5% CO2 and 95% air for 1, 3, and 7 days. The culture medium was changed every 3 days. The MTT solution was prepared by adding thiazolyl blue tetrazolium bromide powder into the phosphate buffered saline (PBS, OXOID, England), and 10 mL of 5 mg/mL MTT solution was added on the first day. The culture dish

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ORIGINAL RESEARCH REPORT

TABLE I. Primer Pairs Used in Real-Time PCR Analysis Gene Gapdh Col1a1 Runx2 Opn

Forward Primer 0

Reverse Primer 0

5 -ACCCAGAAGACTGTGGATGG-3 50 -GAGCGGAGAGTACTGGATCG-30 50 -CCCAGCCACCTTTACCTACA-30 50 -TCTGATGAGACCGTCACTGC-30

was then incubated at 37 C under 5% CO2 and 95% air for 1 day. A total of 100 lL of 10% sodium dodecyl sulphate (SDS, Sigma) in 0.01M hydrochloric acid was added to each well and re-incubated at 37 C in an atmosphere of 5% CO2 and 95% air for overnight. Finally, the absorbance was recorded by a multi-mode detector on the Beckman Coulter DTX 880 at a wavelength of 570 nm with a reference wavelength of 640 nm. The cell viability was determined by the absorbance readings. Cell apoptosis assay. To evaluate the cell apoptosis, flow cytometry (BD FACSCanto II Analyzer) was performed using the Annexin V-FITC/PI kit according to the manufacturer’s protocol (Beyotime, China). The disc-like samples of 13.9 mm in diameter (n 5 6) were placed in 24-well tissue culture plate. A total of 5 3 104 cells/cm2 SaOS-2 human osteoblasts were seeded on each sample and cultured for 1, 3, and 7 days, respectively. The culture medium was changed every 3 days. At respective time points, the cells were collected by using trypsin and were adjusted to the cell density of (2–5) 3 105 cells/mL. The cells were then re-suspended into 195 lL buffer and added 5 lL Annexin VFITC. After incubated at room temperature for 30 min and washed, the cells were re-suspended into 190 lL buffer, and added with 10 lL propidium iodide. Finally, the cells were filtered and analyzed by the flow cytometry. Alkaline phosphatase enzyme (ALP) activity measurement. To determine the osteogenic differentiation behavior, the ALP assay was applied on the Cu-SS sample surfaces. The 317L SS served as control. An aliquot of 1.4 3 104 cells/cm2 mouse MC3T3-E1 preosteoblasts was seeded on sample surfaces for 1, 3, and 7 days at 37 C in an atmosphere of 5% CO2 and 95% air, respectively. The cells were cultured in the DMEM culture medium supplemented with 10% (v/v) fetal bovine serum (Biowest, France), antibiotics (100 U/mL of penicillin and 100 lg/mL of streptomycin), and 2 mM L-glutamine. The culture medium was changed every 3 days. After incubation, the cells were washed with PBS for three times and lysed with 0.1% Triton X-100 at 4 C for 30 min. The cell lysates were centrifuged at 574g under 4 C for 10 min (2–5 Sartorius, Sigma) and 10 lL of the supernatant of each sample was transferred to a 96well tissue culture plate. The ALP activity was determined by a colorimetric assay by using an ALP reagent containing p-nitrophenyl phosphate (Stanbio) as the substrate. The absorbance was recorded by the multi-mode detector on the Beckman Coulter DTX 880 at a wavelength of 405 nm. The ALP activity was normalized to the total protein level

0

5 -CACATTGGGGGTAGGAACAC-30 50 -GTTCGGGCTGATGTACCAGT-30 50 -TATGGAGTGCTGCTGGTCTG-30 50 -AGGTCCTCATCTGTGGCATC-30

of the samples measured by the Bio-Rad protein assay (BioRad). Real-time quantitative RT-PCR analysis. The osteogenic differentiation behaviors of Cu-SS sample were further assessed by real-time quantitative RT-PCR. The relative mRNA expression levels of the commonly used bone markers including type I collagen (Col1a1), runt-related transcription factor 2 (Runx2), and osteopontin (Opn) were measured (primer pairs used are shown in Table I. A total of 1.5 3 105 cells/cm2 mouse MC3T3-E1 preosteoblasts were cultured in the DMEM culture medium supplemented with 10% (v/v) fetal bovine serum (Biowest, France), antibiotics (100 U/mL of penicillin and 100 lg/mL of streptomycin), and 2 mM L-glutamine, and seeded on the samples incubated at 37 C in an atmosphere of 5% CO2 and 95% air for 3 and 7 days, respectively. The culture medium was changed every 3 days. At the respective time points, the total RNA of the cultured osteoblasts was isolated using a TRIZOL reagent (Invitrogen). Chloroform was added to isolate the RNA into the aqueous phase. The upper colorless aqueous phase was transferred to a new 1.5 mL eppendorf and the isopropanol was added to precipitate the RNA. Finally, the RNA pellets were washed with 75% ethanol and dissolved in the RNase inhibitor diethyl pyrocarbonate treated water. The RNA concentrations were determined on the Nanodrop 1000 spectrophotometer (Thermo Scientific). The complementary DNA (cDNA) was reverse-transcribed from 1 mg of total RNA using a high-capacity RNA-to-cDNA master mix kit (Applied Biosystem) by following the manufacturer’s instruction. The real-time PCR was performed on the SYBR Green PCR master mix (Applied Biosystems). The total reaction volume was 25 lL including 12.5 lL 2 3 SYBR Green PCR master mix, 1 lL forward and 1 lL reverse primers, 1 lL cDNA template, and 9.5 lL RNase water. The reaction was performed on the ABI prism 7900HT sequence detection system (Applied Biosystems) and the standard setting with 40 cycles was used to amplify the signal. Finally, the relative mRNA expression level of each gene was normalized to the housekeeping gene glyceraldehyde-3phosphate dehydrogenase (Gapdh) and determined using Ct values. In vivo tests Surgical procedures. A total of 16 two-month old female Sprague-Dawley rats (SD rats) weighing between 200–250 g obtained from the Laboratory Animal Unit of The University of Hong Kong were used for the animal study. The anaesthetic, surgical, and postoperative care protocols were

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FIGURE 2. Cu21 ions released from Cu-SS in physiological saline solution up to 30 days. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 1. (a) Implantations of 317L SS (left) and Cu-SS (right) in the lateral epicondyle of SD rats for 15 days. Black arrows show the implant positions, (b) Set-up of the push-out test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

fulfilled according to the requirements of the University Ethics Committee of The University of Hong Kong and the Licensing Office of the Department of Health of the Hong Kong Government. The rats were anaesthetized with ketamine (67 mg/kg) and xylazine (6 mg/kg) by intraperitoneal injection. The operation sites of the rats were shaved and sterilized, and followed by decortication. One hole, with 2 mm in diameter and 6 mm in depth, was made by a hand driller on the lateral epicondyle by using a minimally invasive approach. Subsequently, the 317L SS and Cu-SS samples of 2 mm in diameter were implanted into the prepared holes on either the right femur of the rats as shown in Figure 1(a), respectively. The wound was then sutured layer by layer and a proper dressing was applied over the incision. After the operation, all the rats received subcutaneous injection of 1 mg/kg terramycin (antibiotics) and 0.5 mg/kg ketoprofen. The rats were euthanized at day 15 postsurgery. New bone formation adjacent to the implants. To monitor the initial change of new bone formation around the samples, the rats were scanned in a Micro-CT device (SKYSCAN 1076, Skyscan Company) according to the designed time points at 3, 7, and 15 days. The CTAn program (Skyscan Company) was applied to examine the Micro-CT datasets for morphometry and densitometry as well as new bone growth adjacent to the implants. In addition, the bone mineral density (BMD) of newly formed bone was measured on the samples implanted for 15 days.

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Biomechanical push-out test. To examine the adhesion strength between the bone tissue and the stainless steel implant, a push-out test was conducted on the bone blocks with Cu-SS and 317L-SS implants harvested from the rat femurs at postoperation 15 days. The setup of the push-out test was shown in Figure 1(b). The pushing rate was set at a rate of 1mm/min on the testing machine (i.e., 858.02 Mini Bionix Material Testing System). The push-out strength could reflect the quality of bone–implant integration. Histological and immunohistochemical analyses. To examine the new bone formation and tissue inflammation, the rats were sacrificed after 3, 7, and 15 days postoperation. The bone samples with implants were harvested and fixed in 10% buffered formalin for 1 day at 4 C and subsequently decalcified in a 0.5M EDTA solution for 2 weeks. The implants were then removed after decalcification, whereas the bone samples were embedded in paraffin and cut into 5 lm thick samples by using the microtone (Leica RM 2135). For histological analysis, the sectioned samples were stained with Giemsa (MEKCK, Germany) stain. The morphological and histological analyses were preformed on the optical microscope to observe the new bone formation and bone–implant integration. In addition, tumor necrosis factor (TNF-a), an inflammatory marker, was used to study the inflammatory response of the implants to the host tissue at days 3 and 7, respectively. Statistics All the in vitro experiments were conducted in triplicate and data from both in vitro and in vivo studies were analyzed by one-way ANOVA. The values were expressed as means 6 standard deviations and statistically significant was considered when p value < 0.05. RESULTS

Estimation of Cu21 ions released from Cu-SS Figure 2 suggested the release pattern of Cu21 ions from Cu-SS sample measured in the physiological saline solution

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TABLE II. Cu21 Ions Concentration Released From Cu-SS After 1–30 Days Immersions in Saline Day/cm2 Cu21 ions (ppb)

1 3

6 12

9 22

22 35

30 45

from day 0 to 30. The release rate was calculated by the slope of the Cu21 ions concentration curve as showing in Figure 2. It indicated that the amount of Cu21 ions release was almost linearly correlated to the immersion time. The average rate of Cu21 ions release from the Cu-SS sample was about 1.4 ppb/day/cm2. The concentrations of Cu21 ions at particular time points were summarized in Table II. Cell morphology and morphometry The enhanced-GFP osteoblasts cultured on the new Cu-SS surface were widely spread at day 1 and 3 as compared with the control shown in Figure 3. After 3 days of culturing, the cells almost reached to 100% confluence on the CuSS sample. Figure 4(a) presented the microscopic view of SaOS-2 human osteoblasts on 317L SS and Cu-SS after 1 day of cell culture, and the aspect ratio and average area of the cells were measured and shown in Figure 4(b,c), respectively. The cells on Cu-SS expressed higher aspect ratio as compared with that on 317L SS surface, indicating that the cells were widely spread on the surface of Cu-SS. In addition, the average cell area on Cu-SS surface was slightly higher than that of 317L SS, although this finding was not statistically significant.

Cell apoptosis Figure 5(a) was the results of apoptosis test with the use of SaOS-2 human osteoblasts cultured on 317L SS and Cu-SS for 1, 3, and 7 days, respectively. Normal cells, apoptotic cells, and necrotic cells were distinguished by using the flow analysis. The measures of apoptotic and necrotic cells on the samples at respective time points were shown in Figure 5(b,c), respectively. Significantly higher percentage of apoptotic cells (p < 0.05) was found on 317L SS sample surface at day 3 and 7. The total amount of necrotic cells found on 317L SS sample surface was also significantly higher (p < 0.05) at day 7, suggesting that the Cu-SS might provide a relatively favorable micro-environment for cells as compared with 317L SS. Moreover, the percentage of normal cells on Cu-SS surface was significantly higher (p < 0.05) in day 3 and 7 [Figure 5(d)], while comparing to 317L SS. Viability and differentiations of osteoblasts Though the cell viability was not significantly different between 317L SS and Cu-SS. The ALP activities of MC3T3E1 preosteoblasts cultured on 317L SS surface in day 3 was significantly lower (p < 0.05) as compared with that of CuSS sample (i.e., 60 U/mg protein for Cu-SS and 46 U/mg protein for 317L SS, respectively). However, the specific ALP activity of the cells on Cu-SS surface at day 7 was mildly higher than that for 317L SS. The findings were shown in Figure 6(b). After verified the osteoblastic differentiation behaviors through ALP assay, the differentiation properties were further assessed by real-time RT-PCR. Figure 6(c) demonstrated the mRNA levels Col1a1, Runx2, and Opn

FIGURE 3. Microscopic views of eGFPOB mouse osteoblasts cultured on 317L SS and Cu-SS for 1and 3 days. 1.7 3 104 cells/cm2 GFPOB were cultured on each sample in 96-well plates. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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FIGURE 4. (a) Microscopic views of SaOS-2 human osteoblasts cultured on 317L SS and Cu-SS for 1 day to evaluate the cell spreading on samples, (b) aspect ratio, and (c) average area of cells on 317L SS and Cu-SS. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 5. (a) Apoptosis analyses of SaOS-2 human osteoblasts cultured on 317L SS and Cu-SS at days 1, 3, and 7 of cell cultures. Percentages of apoptotic cells (b), necrotic cells (c), and normal cells after 1, 3, and 7 days of cell cultures. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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OSTEOGENIC ABILITY OF CU-SS

ORIGINAL RESEARCH REPORT

FIGURE 6. (a) Cell viabilities of SaOS-2 human osteoblasts on 317L SS and Cu-SS using MTT assay. The readings were detected under the absorbance reading at wavelength of 570 nm and a reference wavelength of 640 nm was used to determine the cell viability in comparison to the control, (b) Specific ALP activities of MC3T3-El preosteoblasts cultured on 317L SS and Cu-SS at day 3 and 7. The readings were detected under the absorbance reading at wavelength of 405 nm, and (c) Osteogenic differentiation properties assessed by measuring the mRNA expression level of type I collagen (Col1a1), osteopontin (Opn), and runt-related transcription factor 2 (Runx2) at day 3 and 7. The mRNA level was normalized with the house keeping gene glyceraldehyde 3-phosphate dehydrogenase (Gapdh). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

expressions of MC3T3-E1 preosteoblasts cultured on 317L SS and Cu-SS, respectively. All the gene expressions including Col1a1, Opn, and Runx2 on the Cu-SS sample were significantly higher (p < 0.05) in day 3 as compared with the 317L SS sample. Bone formation ability and bone–implant interfacial strength evaluated by biomechanical push-out test To measure the new bone formation in-vivo, the microcomputed tomography analysis was taken at particular postoperative time points. Figure 7(a,b) displayed the newly formed bone adjacent to the 317L SS and Cu-SS samples implanted to rat femur for 15 days, respectively. Bony tissues were observed on the perimeter of 317L SS and Cu-SS samples after 15 days implantation. The changes of bone volume on 317L SS and Cu-SS samples after 3, 7, and 15 days of implantation were shown on Figure 7(c). It was observed that the bone volume on Cu-SS sample gained 10% increase in day 3 postoperation as compared with that of preoperation, whereas the bone volume on 317L SS decreased by 10% at the same time point. Afterwards, the newly formed bone on Cu-SS sample was likely undergoing remodeling and therefore the total volume of bone gained gradually reduced and then maintained at 4% increment at day 15 eventually. It seems that Cu21 ions released from Cu-SS could trigger the new bone formation. Figure 7(d)

proposed the BMD of newly formed bone around the implants at day 15. The BMD of the bone formed on Cu-SS implant was significantly higher (p < 0.05) than that of the control, implying that the bone on Cu-SS sample was relatively matured and mineralized. Furthermore, the biomechanical push-out test was used to evaluate the quality of bone–implant integration. Figure 7(e) demonstrated the maximum push-out force of 317L SS and Cu-SS samples at 15 days of implantation, respectively. The push-out force found on Cu-SS sample was doubled, while comparing to that of 317L SS (p < 0.05). The results suggested that the Cu-SS implant demonstrated superior bone–implant integration in animal model. Histological and immunohistochemical analyses Figure 8 shows the Giemsa stained bone tissue around the 317L SS and Cu-SS samples at respective time points. After 3 days of implantation as shown in Figure 8(a), new bone started to form on the Cu-SS (green arrows) but not on the 317L SS, in which only large amount of fibrous tissue were found (yellow stars). The amount of newly formed bone continued to increase on the Cu-SS from day 7 to 15 [Figure 8(b,c)]. Although the fibrous tissue layer found on the 317L SS was getting smaller and finally new bone started to grow on it, only a thin layer of newly formed bone was observed after 15 days of implantation. Therefore, this suggested that

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FIGURE 7. (a) Micro-CT images on reconstruction of the lateral epicondyle containing 317L SS and Cu-SS implants after 15 days of postoperation. BMD of the bones around the implants, pointed by yellow arrows, were calculated, (b) Micro-CT 3D reconstruction models of the newly formed bone around 317L SS and Cu-SS after 15 days of postoperation. White color represents the newly formed bone and grey color represents the implants, (c) Change of bone volume around 317L SS and Cu-SS implants throughout the implantation period, (d) BMD of the bones formed around 317L SS and Cu-SS implants after 15 days of implantation, and (e) Average maximum push-out forces of 317L SS and Cu-SS implants after 15 days of implantation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

faster bone formation was found on the Cu-SS samples as compared to the 317L SS. The inflammatory response of the 317L SS and Cu-SS samples to the surrounding tissue was studied by immunohistochemical staining of TNF-a after 3 and 7 days of implantations (Figure 9). A strong positive signal (brown color) was detected largely around the 317L SS on day 3 whereas a significantly low quantity of brown color was found surrounded the Cu-SS samples. Strong signal was still detected on day 7 for the 317L SS. However, the signal diminished around the Cu-SS, which suggested that the release of Cu21 ions from Cu-SS is able to suppress the inflammatory response.

DISCUSSION

Our previous studies demonstrated that the Cu-SS that was fabricated by directly immobilizing Cu during the steel making process could continuously release Cu21 ions to offer the multi-biofunctions to the medical stainless steel. Interestingly, it has been reported that Cu21 ions from Cu2SO4 could stimulate osteoblasts, which were beneficial to the bone tissues. Ewald et al.30 found that Cu21 ions could enhance the cell activity and the proliferation of osteoblasts. Erol et al.31 also found that the bioactive glass-based scaffold coated with alginate cross-linked Cu21 ions could promote the bone regeneration. In the current work, our results showed that compared with the conventional 317L SS, the Cu21 ions released from the Cu-SS obviously promoted the osteogenic differentiation, including the bonerelated gene expressions (Col1a1, Opn, and Runx2) and ALP activity of MC3T3-E1 preosteoblasts, as shown in Figure 6. These results suggest that Cu-SS should possess certain

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ability of osteoinduction, which is important to promote new bone formation.32,33 Osteoinduction means that the undifferentiated and pluripotent cells are somehow stimulated to develop into the bone-forming cell lineages. With the correct stimulus (the inductive agent), an undifferentiated mesenchymal cell can be differentiated into a preosteoblast, a process that constitutes the bone induction.34 Cu21 ions are such a kind of inductive agent. There have been studies showing that Cu21 ions could induce differentiation of mesenchymal stem cells and osteoblastic cells.30,35 Wu et.al.36 incorporated Cu21 ions into a mesoporous bioactive glass (MBG) scaffold, and demonstrated that the Cucontaining MBG scaffold possessed obvious ability of osteostimulation due to the release of Cu21 ions that played a central role to stimulate the ALP activity and the osteogenic gene expression, which is well consistent with results of the current study. Therefore, it can be speculated that Cu21 ions released from the Cu SS should play the key role to stimulate the ALP activity and the osteogenic gene expression of osteoblasts. ALP activity and bone-related gene expression are generally considered to be important indicators of bone differentiation and mineralization.37 Although the mechanism of Cu21 ions enhancing the osteogenic gene expression is still unclear, our results do indicate that Cu-SS should possess obvious ability to stimulate the osteoinduction. The second interesting result is that the Cu-SS promoted the osteoconduction as compared to 317L SS, showing different cell morphology and morphometry (aspect ratio and average area) of osteoblasts when cultured with 317L SS and Cu-SS (Figures 3 and 4), respectively. In the practical situation, osteoconduction depends on the previous osteoinduction, and the bone growth on an implant surface depends on the action of differentiated bone cells.37,38

OSTEOGENIC ABILITY OF CU-SS

FIGURE 8. Histological photographs of Giemsa stained bone tissue formed around the implants after (a) 3 days, (b) 7 days, and (c) 15 days implantations in the lateral epicondyle. (i) Lower magnification (403) pictures showing the whole structure of bones around the implants, whereas (ii) and (iii) higher magnification (1003 and 2003) pictures showing the new bone formations around the implants. ‘I’ represents the implant location. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 9. Immunohistochemical staining of TNF-a of bone tissue around the implants after 3 and 7 days implantations. Brown color represents positive staining and ‘I’ the implant location. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

However, in the case of an implant, the osteoconduction is not only dependent on the condition for bone repair, but also on the implant material and its reaction with surrounding bone tissue. For example, osteoconduction is not possible on certain materials such as silver.3 However, osteoconduction has been seen on metallic biomaterials such as stainless steel.22 Based on the role of osteoinduction due to the Cu21 ions released from Cu-SS surface, it can be suggested that Cu-SS could significantly promote the adhesion and proliferation of bone tissue cultured on its surface, according to the current results including both cell morphology and cell morphometry shown in Figures 3 and 4. Thus, these results demonstrated that the Cu-SS could provide not only the inductive agent of Cu21 ions for osteoinduction but also a better surface for osteoconduction. In addition to the promotion of osteoinduction and osteoconduction, our results showed that Cu-SS could also promote the osseointegration in vivo, an important basis for the application of orthopedic implants, according to the quantitative Micro-CT measurement, qualitative histological observation, and push-out test after the implantation of 317L SS and Cu-SS in animal for 15 days. Osseointegration is not an isolated phenomenon, but instead depends on the previous osteoinduction and osteoconduction. It was found in this study that the Cu-SS could achieve a stable anchorage by a direct bone-to-implant contact test. To begin with, more new bone formation was observed around the Cu-SS implant than the 317L SS from Micro-CT images, 3D reconstruction and histological photographs of Giemsa stained

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bone tissue, as shown in Figures 7 and 8. These results suggested that Cu-SS firstly promoted the new bone formation around it. It has been reported that bone formation would not occur without a proper blood supply.3 Albrektsson39 studied bone remodeling in vivo and came to a conclusion that so-called full vascularization was necessary for the bone formation. The Cu21 ions released from the Cu-SS should be not only a kind of bone inductive agent, but also an agent to promote the vascularization, which has been well proved in previous work.29 Thus, bone tissues would be easily formed around the Cu-SS. Furthermore, the pushout test in this study further showed a more stable bone-toimplant contact for the Cu-SS due to the better formation of new bone around the Cu-SS implant, demonstrating its promotion effect on osseointegration based upon the previous effects of osteoinduction and osteoconduction. Thus, Cu21 ions released from the Cu-SS did have promotion effect on osteogenesis through three successive courses including osteoinduction, osteoconduction, and osseointegration, which are the most important factors to the osteogenic behavior for implants.3 The details can be described that Cu21 ions released from the Cu-SS induce the osteoblast to differentiate, and then promote the growth of osteoblast on its surface, and finally achieve a more stable anchorage with the Cu-SS implant. Figure 10 shows a schematic diagram describing the osteogenic ability of the currently studied Cu-SS. The biocompatibility of a metallic biomaterial should always be the first consideration for clinical application,

OSTEOGENIC ABILITY OF CU-SS

ORIGINAL RESEARCH REPORT

FIGURE 10. Schematic diagram indicating the osteogenic ability of Cu-SS. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

especially for the Cu-SS with certain amount of release of Cu21 ions in this study. Cu is a kind of heavy metal element, which can be detrimental to human health if its amount reaches over a certain level in body. The acceptable daily intake of Cu recommended by the World Health Organization (WHO) is 2–3 mg per day per person.29 For this reason, the release amount of Cu21 ions was measured in this study. The result showed that the daily released amount of Cu21 ions from the Cu-SS was really at trace level of ppb order (Figure 2), which should be rather safe to human health. However, the daily released amount of Cu21 ions after long time such as 3 months still needs to be known, and we will consider this in the future work. Some studies also reported that a relatively lower concentration of Ag1 (LD50 5 3.5 3 1023 mmol/L, about 0.378 ppm) could have strong toxicity to the cells, however, a relatively higher concentration of Cu21 (LD50 5 2.3 3 1021 mmol/L, about 14.72 ppm) still had no toxic effect on the cells.40 That is to say that human body could endure higher amount of Cu21 ions than Ag1 ions. Interestingly, our result of apoptosis analyses showed that Cu-SS had not only lower percentage of apoptotic cells on its surface but also significantly higher percentage of normal cells as compared to the 317L SS, which further demonstrate that the release amount of Cu21 ions should be in the safe range and only beneficial to osteoblasts (Figure 5). In addition to the above data analyses, the biocompatibility of Cu-SS was also seriously evaluated from two aspects, that is, in vitro cytotoxicity and in vivo histocompatibility. From the results of cell viability [Figure 6(a)], the Cu-SS expressed good biocompatibility without cytotoxic effect on osteoblast viability. Furthermore, according to the results of histological analysis (Figure 7), the Cu-SS also expressed better histocompatibility in comparison with 317L SS. Another interesting result was the immunohisto-

chemical analysis of TNF-a, which is a kind of proinflammatory cytokine and reflects the extent of inflammation by its concentration. An increased level of TNF-a has been reported to result in the programmed cell death (apoptosis) and to enhance the phagocytosis of neutrophils undergoing apoptosis.41 In the current study, the immunohistochemical staining of TNF-a on bone tissue after the implantation showed that stronger TNF-a signal was found on 317L SS as compared to Cu-SS, suggesting that Cu-SS could largely reduce the happening of inflammation, which should also enhance the osteogenic ability of Cu-SS (Figure 9). In the study of Suska et al.,42 an increase in the number of leukocytes, a marked decrease of exudate cell viability and the highest total amount of NF-jB were found around Cucoated titanium (Ti) compared with the Ti implant at the same site, which demonstrated the toxic effect and inflammatory response of Cu-coated Ti. However, only 4.5% Cu was immobilized in the present stainless steel (Cu-SS) during the steel making process, which is significantly different from the Cu-coated metallic materials. Compared to the Cucoated Ti, the trace release amount of Cu21 ions (in ppb order) from Cu-SS should not enough to induce the release of cytokines or affinity to RNA and DNA, and finally the toxic and inflammatory response. Therefore, a good biocompatibility of Cu-SS is an important factor as it lays a solid basis for the application of multi-biofunctional Cu-SS implants with osteogenic ability.

CONCLUSIONS

In this study, from both in vitro and in vivo experiments, we successfully demonstrate that the newly developed Cu-SS immobilizing with proper amount of Cu could obviously promote osteogenesis, possess good biocompatibility, and inhibit inflammation due to the sustained release of trace

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2014 VOL 00B, ISSUE 00

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