Osteogenesis stimulation by copper-containing 316L stainless steel via activation of akt cell signaling pathway and Runx2 upregulation

1. Introduction

Metallic biomaterials are widely used as surgical implants for their good mechanical properties, corrosion resistance and biocompatibility. They mainly include stainless steel, cobalt-based alloys, pure titanium and titanium alloys, with stainless steel having the longest history of clinical applications. 316 L stainless steel (316 L SS) is the most extensively used stainless steel among surgical implants, and is recommended to be a priority for manufacturing implants by the American Society for Testing and Materials [1]. Its applications include orthopedic devices such as artificial joints, bone plates, and bone pegs [2]. However, it has stable physical and chemical properties in the human body, leading to a bio-inert performance. Osseointegration with 316 L SS often takes several months to achieve, but fibrous tissue encapsulation after implantation occurs easily.

An effective approach to bone implants is to add beneficial elements such as Ca, Ni, Si, Zn and Sr to improve their osteogenic capabilities [[3], [4], [5]]. Wu et al. [6] developed Cu-containing mesoporous bioactive glass scaffolds, which significantly enhanced the osteogenic differentiation of human bone marrow stromal cells by improving bone-related gene expression including that of alkaline phosphatase (ALP), osteopontin and osteocalcin. From the above results, surface modification through the incorporation of bioactive elements is an effective way to enhance osteogenesis after bone implantation. Indeed, an easier way exists to improve the osteogenic ability of metallic biomaterials. That is, by adding beneficial elements to the metal matrix directly during the metallurgical process, which is a more convenient way to achieve sterilization and processing. Hence such an approach was used in this study.

Cu is not only a widely used alloying element in metals, but also essential in bone growth and development. Although the effect of Cu in bone metabolism is well known, skeletal deformity is always accompanied by a serious Cu deficiency [7]. It has been shown that Cu can stimulate osteogenic differentiation of mesenchymal stem cells, improve the activity and proliferation of osteoblasts, and accelerate collagen deposition [8,9]. Hence a Cu-containing stainless steel should enhance osteogenesis by continuous Cu ions release. In previous studies, Cu-containing 317 L stainless steel was shown to promote osteogenesis in vitro and in vivo [10]. Moreover, Cu also plays an important role in angiogenesis [11], which is a critical process in the recovery of a blood supply for tissues in the periphery of implants. An appropriate amount of Cu ions can stimulate vessel formation through increasing the secretion of vascular endothelial growth factor, the most efficient growth factor for angiogenesis [12]. Cu was shown to enhance the migration and tube formation ability of human umbilical vein endothelial cells (HUVECs) [13]. In addition, infection is recognized as highly detrimental in the clinic and is the main reason for implantation failure [14]. In this regard, Cu is a strong inorganic antibacterial agent. Cu-containing stainless steel was found to possess good antibacterial properties both in vitro and in vivo [15,16]. Hence, Cu looks promising for its use in bone implants with multi-biological functions, including the stimulation of osteogenesis and angiogenesis, as well as having an antibacterial effect.

As an important factor, Cu activated the MAPK pathway in melanoma cells [17], promoted the invasion of prostate cancer epithelial cells by Jagged 1/Notch pathway [18], and protected myocardial damage by up-regulation of pAkt and pGSK-3β [19]. The Akt signaling pathway is one of the most common signaling pathways that has been identified to be implicated in cell proliferation and differentiation [20]. However, whether the Akt signaling pathways is involved in Cu-containing 316 L stainless steel (316L-Cu SS) induced osteogenesis remains to be clarified.

In this work, the osteogenic properties of a 316L-Cu SS was investigated. Relevant mechanisms were analyzed by evaluating and analyzing cell proliferation, apoptosis and attachment, as well as bone-related gene and protein expression by culturing osteoblasts with the steel. It is noted that this study is further deeply focus on the mechanism of promoting osteogenesis of 316L-Cu SS from Akt cell signaling pathway. These results add to an evidence for the clinical application of this novel and promising bone implant material.

2. Experimental

2.1. Materials preparation

Based on the composition of 316L SS, 316L-Cu SS was designed, and both were melted using a 25-kg vacuum induction melting furnace, with chemical compositions listed in Table 1. These casting alloys were then forged at 1100 °C. The metal samples were solution treated at 1050 °C for 0.5 h, followed by a water quench. Metal samples with two sizes, Φ10 mm × 1 mm and Φ5 mm × 1 mm, were prepared for different tests. Smaller metal samples were only used for the CCK-8 assay. All the samples were grinded with SiC sand papers up to grade 2000# and the roughness is around RA 250 nm, soaked in absolute ethyl, cleaned with supersonic and deionized water, and finally sterilized at 121 °C prior to experiments.

2.2. Cell culture

Human fetal osteoblasts (hFOB 1.19) purchased from the American Type Culture Collection were used in this study. Cells were cultured in high glucose Dulbecco’s modified Eagle medium (DMEM; Hyclone, Logan UT, USA) supplemented with 10% fetal bovine serum (Gibco/Thermo Fisher Scientific, Waltham, MA, USA), 80 U/mL penicillin (Hyclone), and 0.08 mg/mL streptomycin (Hyclone).The medium was changed every 2 days. When cells reached 80% confluence, they were sub-cultured at a ratio of 1:2 and incubated at 37 °C in a humid atmosphere containing 5% CO₂.

2.3. CCK-8 assay

A Cell Counting Kit (CCK)-8 assay was performed to evaluate the effect of 316L-Cu SS on cell proliferation. Metal samples with a size of 5 mm × 1 mm were put into 96-well plates and 100 μL of medium containing 5 × 10³ cells was added to each well. The medium was removed and replaced by a 110 μL solution composed of medium and CCK-8 solution (Dojindo, Kumamoto, Japan) at a ratio of 10:1 on days 1, 3 and 7, respectively. After incubation at 37 °C for 2 h, a 100 μL solution was transferred to a fresh 96-well plate. The absorbance at 450 nm was measured by a microplate reader (Thermo Scientific Multiskan). All results were processed after background subtraction.

2.4. Flow cytometry

Cell apoptosis was evaluated using annexin V-FITC/propidium iodide (PI) double staining apoptosis detection kit (Vazyme Biotech, Nanjing, China) and flow cytometry. Cells (3 × 10⁴/well) were seeded on the surface of each sample in 48-well plates. At the indicated time, cells in every fourth well were collected as one sample using EDTA-free trypsin. Then cells were twice washed with cold phosphate buffered saline (PBS). Binding Buffer (1×, 100 μL) was added to re-suspend cells. Next, 5 μL of annexin V-FITC and 5 μL of PI solution were added for staining. The mixed solution was incubated in the dark for 10 min and supplemented with 400 μL 1 × Binding Buffer before detection. Stained cell samples were measured by flow cytometry within 1 h and analyzed using FlowJo7.6.

2.5. Cell morphology

Cell morphologies on the surfaces of samples were observed by detecting filamentous actin (F-actin) of the cytoskeleton using immunofluorescence. Medium (100 μL/well) containing 2.5 × 10⁴ cells was added to the surface of a metal sample in each well of a 24-well plate. Several hours later, 1 mL of medium was added per well till cells were adherent. After incubation for 4 h or 24 h, cells were washed three times with warm PBS, fixed in 4% paraformaldehyde for 15 min, and then permeabilized with 0.5% Triton X-100 for 5 min. Phalloidin-FITC (Sigma-Aldrich, St Louis, MI, USA) at a concentration of 5 μg/mL was incubated with cells for 1 h at 37 °C in the dark. DAPI (Beyotime, Jiangsu, China) was then used to stain cell nuclei. An inverted fluorescence microscope was used to observe results.

2.6. RT-qPCR

Osteogenesis related gene expression in cells cultured on the surface of metal samples was studied by reverse transcriptase quantitative PCR (RT-qPCR). Cells (3 × 10⁴) were seeded on metal samples in 48-well plates. At the indicated time, cells in every third well were collected as one sample, and total RNA was extracted by TRIzol (Vazyme). The concentration of RNA was measured using a nano-drop 1000 reader and then adjusted to 150 ng/μL. Copy DNA from a 3 μL RNA solution was synthesized using a TaKaRa PrimeScript™RT reagent Kit (TaKaRa, Kutsatsu, Japan). The product was then diluted by 10 μL double-distilled water, 2 μL of which was mixed with 10 μL SYBR Premix, 6.4 μL dH₂O, and 1.6 μL primers at a concentration of 10 μM to prepare the reaction solution according to TaKaRa SYBR^® Premix Ex Taq™ II (TliRnaseH Plus). GAPDH was used as a housekeeping gene. All primers were designed based on information in the National Center for Biotechnology Information database and confirmed by Primer-BLAST. Sequences for collagen I, Runx2 and GAPDH are listed in Table 2. Relative expression levels for each gene were calculated by the formula, 2^-ΔΔCT.

2.7. Western blotting

Western blotting was performed for the direct detection of Runx2, pAkt and total Akt proteins. Cells (3 × 10⁴) were seeded on the metal samples, with or without 10 μM Akt inhibitor (GSK2141795), in 48-well plates. On day 3, cells in every 20th well were collected as one sample. Total proteins from cells were extracted by mammalian protein extraction reagent (M-PER; Thermo Scientific). After the removal of tissue debris by centrifugation of 800 ×g at 4 °C for 15 min, the supernatants were used for western blotting analysis. Samples, each containing a total of 60 μg protein, were loaded and separated on 4%-12% gradient mini gels (Thermo Fisher Scientific) and electrotransferred onto polyvinylidene difluoride membranes (Thermo Fisher Scientific). The membranes were blocked with 5% milk powder (BD Bioscience, San Jose, CA, USA), washed with tris-buffered saline with Tween 20 (TBST), and incubated with rabbit polyclonal anti-Runx2 antibody (1:500; Proteintech, Rosemont, IL, USA), rabbit polyclonal anti-Ser⁴⁷³-phosphorylated-Akt antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-Akt antibody (1:1000; Cell Signaling), and rabbit polyclonal anti-GAPDH antibody (1:5000; Proteintech) overnight at 4 °C. The membranes were washed with TBST and incubated with peroxidase-conjugated anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as a secondary antibody. The membranes were washed with TBST, and developed using Super Signal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, IL, USA). The protein signal was imaged and analyzed using Image lab 4.1 (BioRad, Hercules, CA, USA).

2.8. Statistical analysis

All experiments were performed in triplicate and data were expressed as mean ± standard deviation. An independent-sample t test in SPSS was used to analyze statistical differences. A p-value of less than 0.05 was considered to be statistically significant.

3. Results and discussion

3.1. Cytotoxicity of hFOB cultured with stainless steel

It is well known that excess Cu ions are toxic due to the generation of reactive oxygen species via Fenton or Haber-Weiss reactions [21]. Thus it was essential to examine the cytotoxicity of 316L-Cu SS. In our previous study, daily Cu releasing amount from the 316L-Cu SS within 7 days were measured. The results demonstrated that the release amount of Cu concentration of each time point is significantly higher than that of 316L SS, and all the values were maintained at the range of 6.5 to 8.5 μg/L [22]. To study the effect of Cu addition on the cytotoxicity of hFOB, cells were cultured on the surface of stainless steel for 1, 3 and 7 days. As shown in Fig. 1, the number of cells grew with time, but a significant difference between 316L SS and 316L-Cu SS was not noted within 7 days. Because of good corrosion resistance, the amount of Cu ions released from 316L-Cu SS is bio-safe and showed no cytotoxicity. Many studies claimed that an appropriate dose of Cu ions could enhance the proliferation of mesenchymal stem cells or osteoblasts [7,22,23]. However, our result showed that 316L-Cu SS did not release enough Cu ions to promote osteoblast proliferation.

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Fig. 1.   Cell Counting Kit (CCK)-8 proliferation assay of human fetal osteoblasts (hFOB) cultured on 316L stainless steel (316L SS) and copper-containing 316L stainless steel (316L-Cu SS) for 1, 3 and 7 days, respectively (p > 0.05).

3.2. Apoptosis of hFOB cultured with stainless steel

Cell apoptosis is a programmed death that keeps a dynamic balance of cell numbers within the body. It becomes activated on the initiation of an immune response or when toxic substances invade the body [24]. To further analyze its influence on cell growth, cell apoptosis on the surface of stainless steel for 1 and 3 days was studied by flow cytometry. As shown in Fig. 2, on day 1, the apoptotic rate of cells on 316 L SS was (10.8 ± 3.8)%, while that of cells on 316L-Cu SS was (14.3 ± 3.6)%. Therefore, Cu ion release stimulated cell apoptosis at an early stage. However, the apoptotic rate of cells on 316L-Cu SS was lower than that of cells on 316 L SS by day 3. For bone implants, apoptosis occurs frequently at sites of active bone remodeling. In repairing a bone defect, bone resorption is achieved by nearby osteoclasts, and bone formation is then completed by osteoblasts. The release of mineral ions at an early stage induces the apoptosis of osteoblasts to create a balance between bone formation and resorption [25]. It is coincidental that 316L-Cu SS promoted osteoblastic apoptosis on day 1; as co-culture time increased to 3 days, however, the opposite was the case. Hence, hypothetically speaking, the effect of 316L-Cu SS on osteoblastic apoptosis is incorporated within the procedure of bone remodeling.

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Fig. 2.   Apoptosis of human fetal osteoblasts (hFOB) cultured on 316L stainless steel (316L SS) and copper-containing 316L stainless steel (316L-Cu SS) for 1 and 3 days, respectively (p > 0.05).

3.3. Cell morphology on stainless steel surfaces

The cytoskeleton plays an important role in many cellular activities, such as attachment, migration, and mitosis, to name a few. At the onset of mitosis, F-actin relocates from the edge of cell to form an equatorial ring, and then actin and myosin slide to pull the plasma membrane inward to divide the cell and complete mitosis [26]. The amount of polarized F-actin is proportional to migration ability, because it is the main component in a pseudopodium. F-actin stains of the cytoskeleton of hFOB cultured on surfaces of stainless steel for 4 h and 24 h are shown in Fig. 3. At 4 h, cells on 316L-Cu SS covered a markedly larger area. After culture for 24 h, cells on both materials grew well. However, more and longer pseudopodia were present in cells grown on the surface of 316L-Cu SS. Hence F-actin staining highlighted how osteoblasts on the surface of 316L-Cu SS showed increased mitosis and migration, which would increase the interaction between implants and tissue.

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Fig. 3.   Morphologies of human fetal osteoblasts (hFOB) cultured on 316L stainless steel (316L SS) and copper-containing 316L stainless steel (316L-CuSS) for 4 h and 24 h.

Taken together, the biocompatibility of 316L-Cu SS, including cytotoxicity, cell apoptosis and cell morphology, was examined. The CCK-8 assay showed that 316L-Cu SS had non-cytotoxic effect when co-cultured hFOB within 7 days. However, this material promoted cell apoptosis at an early stage while inhibiting it at a later stage. The Cu-containing surface of the steel was found to be more suitable for hFOB to attach and spread. Cultured hFOB on a Cu-containing steel surface showed better attachment and growth.

3.4. Bone-related gene expression

The extracellular matrix is an important part of bone and cartilage, which is mainly composed of collagen and proteoglycans. Its metabolism is bound up with the fracture-healing process. The gene expression of collagen induces bone-like tissues and forms a bone matrix through mineralization to recover the normal structure and function of bone. Cu ions are known to stimulate collagen deposition [27]. ALP is an important marker in osteoblastic differentiation, which can regulate the mineralization process by producing free phosphate [28]. Runx2 is a transcription factor controlling skeletal development through regulating chondrocyte and osteoblast differentiation [29]. Runx2 expression is regulated by the canonical WNT signaling pathway and it is a target of β-catenin/T-cell factor (TCF)-1 in the stimulation of bone formation [30].

The bone-related gene expression (collagenI, ALP and Runx2) of hFOB cultured on the surfaces of 316L SS and 316L-Cu SS for 1, 3 and 7 days are shown in Fig. 4(a-c). For collagen I, its relative mRNA expression was significantly improved when cells were grown on 316L-Cu SS at all indicated times (Fig. 4(a)). The difference in values between cells grown on 316 L SS and 316L-Cu SS gradually increased with incubation time. For ALP, 316L-Cu SS showed no effect on day 1, but its gene expression was enhanced significantly on days 3 and 7 (Fig. 4(b)). 316L-Cu SS also significantly stimulated the mRNA expression of Runx2 on days 1, 3 and 7 (Fig. 4(c)).

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Fig. 4.   Results of reverse transcriptase-quantitative PCR (RT-qPCR). Relative mRNA expression of collagen I (a), ALP (b) and Runx2 (c) in human fetal osteoblasts (hFOB) cultured on 316L stainless steel (316L SS) and copper-containing 316L stainless steel (316L-Cu SS) for 3 days. #p < 0.05 compared with 316L SS.

3.5. Protein expressions of Runx2, pAkt and Akt

In various cell culture systems, PI3K-Akt signaling has been implicated as a critical pathway for the differentiation of skeletal component cells including chondrocytes, osteoblasts, myoblasts, and adipocytes. In addition, there is substantial evidence that the Akt signaling pathway is essential for osteogenesis in vitro [31,32]. Further, bone development is severely delayed in mice lacking both Akt1 and Akt2 [33]. Runx2 and Akt signaling are mutually dependent on each other in the regulation of osteoblast differentiation and migration [34]. However, the relationship between Runx2 and Akt signaling is not known in hFOB.

After the co-culture of cells on surfaces of stainless steel, with or without 10 μM of a Akt inhibitor, GSK2141795, for 3 days, pAkt, Akt and Runx2 protein expression was detected by western blotting. As shown in Fig. 5, the ratio of pAkt/Akt on 316 L SS was (1.14 ± 0.05), while that on 316L-Cu SS was (1.46 ± 0.08); thus, 316L-Cu SS upregulated the phosphorylation of Akt significantly. GSK2141795 inhibited the copper-induced phosphorylation of Akt significantly, and reduced the ratio of pAkt/Akt (0.93 ± 0.06). The relative intensity of Runx2 in cells grown on 316 L SS was 0.48 ± 0.02, while that of cells grown on 316L-Cu SS was 0.57 ± 0.04. Hence 316L-Cu SS significantly upregulated Runx2 expression, and GSK2141795 reduced the intensity of Runx2 to 0.40 ± 0.03 (Fig. 5).

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Fig. 5.   Effect of Akt inhibitor (GSK2141795) on copper-containing 316L stainless steel (316L-Cu SS) induced osteogenesis. Protein expression of Runx2, phospho-Akt and Akt (A); the ratio of phospho-Akt to total Akt (B) and Runx2 to GAPDH (C) were assessed by western blotting. (^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001).

Taken together, the bone-related gene expression including Runx2, ALP, collagen I was upregulated via activation of the Akt/Runx2 pathway on hFOB.

4. Conclusion

316L-Cu SS is a novel metallic material that has potential to be used in orthopedic implants to improve osteogenesis by Cu ion release. The present study demonstrated that 316L-Cu SS was bio-safe and behaved no cytotoxicity in comparison with 316L SS. 316L-Cu SS increased cell apoptosis on day 1 and decreased it on day 3, which may cooperate with the procedure of bone remodeling. Human FOB attached more quickly and showed better spread on the surface of 316L-Cu SS than 316L SS. 316L-Cu SS also significantly enhanced the gene expression of collagen I, ALP and Runx2. The protein expression of Runx2 was stimulated by 316L-Cu SS via an Akt cell signaling pathway (Fig. 6). The above results indicate that 316L-Cu SS is a promising agent to accelerate osteogenesis in orthopedic implants.

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Fig. 6.   Schematic diagram about “Osteogenesis Stimulation by Copper-containing 316 L Stainless Steel via Activation of Akt Cell Signaling Pathway and Runx2 Upregulation”.

Acknowledgements

This work was financially supported by the National Key Research and Development Program of China (No. 2018YFC1106600), the National Natural Science Foundation (Nos. 81571778 and 51631009) and the Science and Technology Plan of Shenyang (Nos. 17-230-9-42 and 18-014-4-28).