Evaluation of the Size-Dependent Cytotoxicity of DLC - American

Article pubs.acs.org/journal/abseba

Evaluation of the Size-Dependent Cytotoxicity of DLC (Diamondlike Carbon) Wear Debris in Arthroplasty Applications T. T. Liao, Q. Y. Deng, S. S. Li, X. Li, L. Ji, Q. Wang, Y. X. Leng,* and N. Huang Key Laboratory for Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, Sichuan 610031, China ABSTRACT: Patients with DLC (diamond like carbon)-coated artificial joints may be exposed to a wide size range of DLC wear debris (DW). In this study, the cytotoxicity of DW of different size ranges (0−0.22, 0.22−0.65, 0.65−1.0, and 1.0−5.0 μm) was evaluated. The microstructure and physical characteristics of DW were investigated by Raman spectroscopy, transmission electron microscopy (TEM), scanning electron microscope (SEM), and dynamic light scattering (DLS). Macrophages, osteoblasts, and fibroblasts were incubated with DW of different size ranges respectively followed by cytotoxicity evaluations of inflammatory cytokines, alkaline phosphatase (ALP) assays, and related signal protein expression analysis. The results showed that, except for the size range of 0−0.22 μm, DW cytotoxicity showed a size-dependent (0.22−5.0 μm) decrease with increasing size. Within the range of 0.22−5.0 μm, DW of larger size resulted in lessened inflammatory response and enhanced osteoblastogenesis and fibrogenesis, with increased viability of cells (macrophages, osteoblasts, and fibroblasts), better morphology, less release of pro-inflammatory factors and more release of anti-inflammatory factors. The results demonstrated that DW sizes below 0.22 μm had less negative effects on cell adhesion and growth because of the BSA (bovine serum albumin) encapsulation effect. These findings provide valuable knowledge about the comprehensive mechanism of promotion of inflammatory response and inhibition of osteoblastogenesis and fibrogenesis induced by DW. In conclusion, an effective system of biocompatibility evaluation for different sizes of DW was derived. KEYWORDS: DLC wear debris, cytotoxicity, size, macrophages, osteoblasts, fibroblasts

1. INTRODUCTION Total hip arthroplasty (THA) is considered the most effective management strategy for end-stage osteoarthritis.1 Because of their good load-bearing capability and wear resistance, metalon-metal (MoM) prostheses have become the primary bearing material in artificial joints.2 However, metal debris and ions generated from MoM prostheses could cause tissue necrosis and inflammation, ultimately leading to aseptic loosening and implant failure.3,4 To decrease the release of metal wear debris and ions, researchers have incorporated surface modifications on the MOM implant.5−7 Because of its excellent corrosion resistance, wear resistance, and biocompatibility, diamondlike carbon (DLC) has been the most promising candidate for surface modification.8,9 After DLC modification, DLC wear debris (DW) is still generated and released into the joint interface.10 DLC particleinduced osteolysis is a major cause of aseptic loosening in total hip replacement, and the osteolytic potential of wear debris is dependent on the particle size.11 However, little is known about the mechanisms underlying the toxicity of differentially sized DW. Numerous reports have indicated that various kinds of conventional wear particles induce different cellular and molecular responses at the periprosthetic site in a sizedependent manner.12−14 For instance, Aiqin Liu et al.15 © XXXX American Chemical Society

reported that ultra high molecular weight polyethylene (UHMWPE) wear particles under 50 nm had no effect on inflammatory response. Marco S. Caicedo et al.16 found that the irregular cobalt−chromium−molybdenum (CoCrMo) wear debris of a larger size range induced more inflammatory reactivity when compared to an equal dose (particles/cell) of smaller/spherical CoCrMo particles in vitro. Therefore, cytotoxicity evaluation of DW of different sizes should be performed. DW is associated with osteolysis and even implantation failure of DLC-coated artificial joint implants. First, DW of different sizes is shown to the interface of joint prosthesis. Macrophages may be activated by the exposure of DW, which is accompanied by the secretion of pro-inflammatory factors, such as TNF-α (tumor necrosis factor-α) and interleukins (IL-1b, IL-6, and IL-8).17,18 The amount of pro-inflammatory factors reflects the degree of inflammatory response, and it also trigger a series of variation in interface surroundings.19,20 After that, osteoblast activities decrease and osteoclast activities increase, resulted in bone resorb at the interface tissue of joint prosthesis. Received: October 7, 2016 Accepted: March 16, 2017 Published: March 16, 2017 A

DOI: 10.1021/acsbiomaterials.6b00618 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Additionally, DW was then resuspended in 1 mL of phosphate buffered saline (PBS) and completely boiled. The DW suspension was stained through coomassie brilliant blue and measured at 450 nm by the enzyme standard instrument (Synergy H1, USA). Another method to evaluate adsorption amount was FITC (fluorescein isothiocyanate) decoration. First, 0.1 mg DW was dispersed in 0.5 mL PBS with a 15 min sonication and collected on clean glass sheets. Then, FITC-decorated BSA was evaluated via the adsorption morphology by fluorescence microscopy (Olympus IX 51, Japan). 2.4. Evaluation of Inflammatory Reaction. RAW 264.7 murine macrophages were mechanically dispersed in Dulbecco’s modified Eagle’s medium (DMEM, Corning cellgro@, USA) supplemented with fetal bovine serum (FBS, 10%), followed by seeding of 2 × 104 cells/well and 24 h cultivation. Then, 1 mL/well suspensions of different sized particles (0−0.22, 0.22−0.65, 0.65−1.0, and 1.0−5.0 μm) were added to the wells and the cells were incubated at 37 °C in a 5% CO2 humidified atmosphere for 12 and 24 h. 2.4.1. Macrophage viability assays. The viability of the macrophages was investigated by CCK-8 after 12 and 24 h of culture. At each time point, the culture medium was removed. Then, 0.35 mL/well fresh culture medium containing 10% CCK-8 solution was added and incubated with the samples for 4 h. Finally, 0.2 mL/well of incubated medium was transferred to a 96-well plate for OD measurement at 450 nm by a microplate reader (Synergy H1, USA). 2.4.2. Macrophage Morphology. Samples were rinsed three times with PBS, fixed with 2.5% glutaraldehyde overnight at 4 °C, and then the fixed samples were again rinsed three times with PBS. Afterward, samples were dehydrated in a graded ethanol series (50, 75, 90, and 100% vol/vol) for 15 min each. Then, samples were put in the fume hood overnight to ensure completely evaporation. Morphology of the macrophages was assessed by scanning electron microscopy (SEM, Quanta 250, FEI, USA). 2.4.3. Cytokine Analysis. The supernatants from the samples were analyzed for their cytokine expression levels. The supernatant of each sample was collected and stored at −20 °C until cytokine analysis. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), MMP-2, MMP-9, and tissue inhibitor of metalloproteinases-1 (TIMP-1) were determined using commercial sandwich enzyme-linked immunosorbent assay(ELISA) kits (Boster, China), following the instructions of the manufacturer. 2.5. Primary Mouse Osteoblast Culture and Evaluation. Osteoblasts were cultured in Minimum Essential Medium Alpha (αMEM, Corning cellgro@, USA) supplemented with 10% FBS. The osteoblast monolayers were trypsin-digested and the cells were adjusted to 1 × 104 cells/mL with culture medium. Then, 1 mL/ well cell suspension was seeded onto the DW samples and incubated at 37 °C with 5% CO2. 2.5.1. Osteoblast Proliferation by CCK-8. The proliferation of the osteoblasts was investigated using CCK-8 after 1 and 3 days of culture. The specific procedures were the same as those for the viability evaluation of macrophages. 2.5.2. Osteoblast Morphology by Immunofluorescent Staining. Osteoblast morphology was evaluated by fluorescence microscopy (Olympus IX 51, Japan) after staining with rhodamine 123 (Sigma USA) and 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Sigma USA) at 1 and 3 days. 2.5.3. Alkaline Phosphatase (ALP) Activity Analysis. The effect of different sizes of DW on osteoblast differentiation was determined by assessing ALP activity. After 1 and 3 days of culture in 24-well plates, the supernatant and lysis solution were collected and analyzed for ALP activity. The ALP activity was measured using double distilled water as a substrate, and the level of ALP was determined using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, China), following the instructions of the manufacturer. 2.5.4. Expression of OPG and RANKL. In order to evaluate the related signal proteins in osteoblastogenesis response, after 24/72 h cultivation, osteoblast supernatants were collected and osteoblast monolayers were lysed to extract the osteoblast-associated proteins, i.e., OPG and RANKL, which were measured by ELISA kits. Reported

The loss of bone metabolism lead to aseptic loosening of the prosthesis even the failure of implant. Varying sizes of DW may trigger other harmful biological effects in the interface of artificial joints, such as osteoblastogenesis and fibrogenesis. It is well-known that many different signal paths contribute to this biological process. The function of matrix metalloprotease-2 (MMP-2) and matrix metalloprotease-9 (MMP-9) is the degradation of extracellular matrix, and the tissue inhibitor of metalloproteinase-1 (TIMP-1) could prevent the activities of MMPs. The ratio of MMPs/TIMPs could reflect the joint response to DW exposure.21,22 Furthermore, in the RANKL/ RANK/OPG signal pathway, RANKL (receptor of activator of nuclear factor kB-ligand) promotes bone resorption by interacting with the receptor activator of nuclear factor κB (RANK) on osteoclasts to favor osteoclast differentiation and activation. OPG (osteoprotegerin) blocks bone resorption by preventing RANKL-RANK interaction via competitive binds to RANKL. The OPG/RANKL ratio could be a useful metric for evaluating osteolysis.23 Nevertheless, the mechanisms of how the comprehensive system reacts to DW is still unknown. Thus, comprehensive evaluation of the cyto- and biocompatibility of DW of different sizes is necessary to further develop DLC film for in vitro or in vivo biomedical applications.24,25 In the present study, we prepared DW with varying sizes using filter membranes and evaluated the equality by testing their material characteristics. Later, we systemically performed cytotoxicity evaluation of varying sizes of DW through analysis of cell viability and related factor expression (TNF-α, IL-6, IL10, MMP-2, MMP-9, TIMP-1, OPG, and RANKL). This research builds on previous findings by exploring the hypothesis that various sizes of DW (0−0.22, 0.22−0.65, 0.65−1.0, and 1.0−5.0 μm) may distinctly influence the viability and activation/differentiation of macrophages/osteoblasts/fibroblasts and participate in the process of aseptic prosthetic loosening.

2. MATERIALS AND METHODS 2.1. Preparation of DW of Different Sizes. DLC films were deposited by filtered cathodic vacuum arc (FCVA) deposition26 at a direct current bias voltage of 80 V. Glass sheet were used as substrate. Then, samples were sonicated for 5 min with distilled water, and lapping using an agate mortar was performed for 9 h. We then collected the DW suspension and filtered it through different sizes of filter membranes. There were 4 sizes of filter membranes (0.22 μm, 0.65 μm, 1.0 μm, and 5.0 μm) used to prepare four size ranges (0− 0.22 μm, 0.22−0.65 μm, 0.65−1.0 μm, 1.0−5.0 μm) of DW. At last, DW was centrifuged and dried, and a 10 μg/mL particle suspension was prepared for cell experiments. In addition, DW samples were first prepared with a 15 min sonication, hatched overnight (>4 h) at 4 °C, and finally sonicated for another 15 min before applying them to cultured cells to evaluate cytotoxicity. 2.2. Characteristics of DLC Wear Debris. Size and shape of DW were characterized by JEM-2100F field emission transmission electron microscope (TEM, JEOL, Japan) and scanning electron microscope (SEM, JSM-7001F, JEOL, Japan). Size distribution and zeta potential of DW were determined by dynamic light scattering (ZETA-AIZER, Malvern, UK). Raman spectra from 600 to 2000 cm−1 were obtained using a Raman spectrometer (Renishaw Invia, UK) activated by a 514 nm Ar+ laser (with 20 mW power, 2 μm diameter spot diameter). 2.3. Protein Adsorption. BSA (bovine serum albumin) adsorption was evaluated by the method of Coomassie brilliant blue. First, 0.1 mg of DW was dispersed in 0.5 mL of PBS with a 15 min sonication. Then, DW suspension samples were mixed with 1 mL of BSA (2 mg/mL protein in PBS) solution through another 5 min sonication. After incubation at 37 °C for 48 h, BSA-adsorbed DW was isolated from BSA solution by centrifugation at 3,000 rpm for 15 min. B


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Figure 1. (a) SEM, (b) TEM, (c) size range, and (d) Raman spectrum evaluation of material characteristics of DW.

band site. The ID/IG value was calculated by fitting two Gaussian curves and is also listed in Table 1. The ID/IG values for DW with the size ranges of 0−0.22, 0.22−0.65, 0.65−1.0, and 1.0−5.0 μm are around 1.3, showing graphitization characteristics and similar structure. Table 2 shows the mean size and zeta-potential of DW of different sizes. The mean sizes of 0−0.22, 0.22−0.65, 0.65−1.0,

levels of OPG and RANKL produced by osteoblasts are their summation from lysates and supernatants. Additionally, the ELISA results were normalized by the protein amount determined by the microplate reader. 2.6. Fibroblast Culture and Evaluation. Mouse embryo fibroblasts (3T3) were mechanically dispersed with DMEM (Corning cellgro@, USA) supplemented with FBS (10%), seeding at a density of 8 × 103 cells per well, and the cells were incubated at 37 °C in a 5% CO2 humidified atmosphere for 1 and 3 days. 2.6.2. Fibroblast Viability. Fibroblast viability was determined by CCK-8 at 1 and 3 days. The procedures was the same as that described previously for the macrophage assessment. 2.6.3. Fibroblast Morphology. Fibroblast morphology was determined by rhodamine 123 at 1 and 3 days. The specific procedures was the same as that described previously for the osteoblasts. 2.7. Statistical Analysis. One-way ANOVA was conducted using the IBM SPSS Statistics 19 software, and data are reported as mean ± standard error (n = 3). In this study, p < 0.05 was the threshold of significance. In the figures, extremely significant differences (p < 0.001) and significant differences of p < 0.01 and p < 0.05 are denoted with ***, **, and *, respectively.

Table 2. Mean Size and Zeta-Potential of DW of Different Sizes

Table 1. Structural Characterization from Gaussian-Fitted Raman Spectrum of DW of Different Sizes D band (cm−1)

G band (cm−1)

ID/IG

0−0.22 0.22−0.65 0.65−1.0 1.0−5.0

1362 1375 1367 1353

1581 1583 1585 1575

1.2 1.5 1.3 1.3

mean size (μm)

0−0.22 0.22−0.65 0.65−1.0 1.0−5.0

0.133 0.315 0.755 2.783

zeta-potential (mV) 16.13 −9.77 −8.48 17.27

± ± ± ±

0.25 1.11 0.16 0.81

and 1.0−5.0 μm DW are 0.133, 0.315, 0.755, and 2.783 μm, respectively, which correspond with the size range results shown in Figure 1. The zeta-potentials of 0−0.22, 0.22−0.65, 0.65−1.0, and 1.0−5.0 μm samples are 16.13 ± 0.25, −9.77 ± 1.11, −8.48 ± 0.16, and 17.27 ± 0.81 mV, respectively. These data indicate that four size ranges (0−0.22, 0.22−0.65, 0.65− 1.0, and 1.0−5.0 μm) of DW was successfully prepared. 3.2. Protein Adsorption. Figure 2 shows the BSA adsorption of DW of different sizes. As shown, the fluorescence of 0−0.22 and 1.0−5.0 μm DW is greater than that of 0.22− 0.65 and 0.65−1.0 μm (Figure 2a) DW, and the OD values of 0−0.22 and 1.0−5.0 μm DW are higher than those of 0.22− 0.65 and 0.65−1.0 μm (Figure 2b). This indicates that 0−0.22 and 1.0−5.0 μm DW show an obviously higher BSA adsorption than samples of 0.22−0.65 and 0.65−1.0 μm. 3.3. Inflammation Response. Figure 3 shows the macrophage viability after exposure to different sizes of DW at 12 and 24 h, as determined by the CCK-8 assay. At both 12 and 24 h, the macrophage viability of the control is significantly higher than that of the other samples, and 0−0.22 and 1.0−5.0 μm DW-exposed macrophages have considerably higher viability than 0.22−0.65 and 0.65−1.0 μm DW-exposed macrophages. Although there is no significant difference between macrophages exposed to 0.22−0.65 μm and 0.65− 1.0 μm sizes at 12 h, macrophage viability in the 0.22−0.65 μm group is significantly lower than that of the 0.65−1.0 μm group

3. RESULTS 3.1. Characterization of DW. Figure 1 shows the material characteristics of DW of in different sizes. Figure 1a , b shows the SEM and TEM images of DW, respectively. DW displays a polygon shape and the size is just within the four size ranges (0−0.22, 0.22−0.65, 0.65−1.0, and 1.0−5.0 μm). Figure 1b also shows the diffraction spot of DW. All the DW of different sizes are in an amorphous state. Figure 1c shows the size range of DW, which is matched the results of SEM and TEM imaging. Table 1 shows the results from Gaussian-fitted Raman spectrum of DW of different sizes. The Raman results show that all the DW samples of different sizes have a similar G/D

samples (μm)

samples (μm)

C


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Figure 2. Adsorption of BSA of different sizes of DW, including (a) fluorescent images and (b) OD values.

for DW in the 0−0.22 μm range, macrophage viability after exposure to DW increase with increasing size. Figure 4 shows macrophage morphology determined by SEM. For macrophages cultured without particles, there is almost no pseudopodia extending (round shape) from the cells. Within size range of 0.22−5.0 μm DW, macrophages increase in number and have a more round shape. After exposure to DW of 0.22−0.65 μm, most of the macrophages have totally extended pseudopods. These data suggest that except for DW in the 0−0.22 μm range, macrophages after exposure to DW show a size-dependent deformation, and less deformation with increasing size. TNF-α and IL-6 are typical pro-inflammatory cytokines and IL-10 is an anti-inflammatory cytokine released by macrophages.27,28 Figure 5 shows the release of inflammatory cytokines at 12 and 24 h. Figure 5a displays the release of TNF-α after 12/24 h culture. Except for the 0−0.22 μm DW group, macrophages release smaller amounts of TNF-α with increasing sizes of DW (0.22−5.0 μm) at 12 h. However, there is no significant difference among the DW sizes 0−0.22, 0.22− 0.65, and 0.65−1.0 μm at 24 h. In addition, macrophages after

Figure 3. Macrophage viability after exposure to different sizes of DW at 12 and 24 h, as determined by the CCK-8 assay.

at 24 h. Except for samples exposed to 0.22−0.65 μm DW, macrophage viability in all the samples at 24 h is significantly higher than the viability at 12 h, indicating apparent proliferation from 12−24 h. These data suggest that, except

Figure 4. SEM images of macrophages challenged with different sizes of DW at 24 h. D


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tory response through a negative feedback mechanism, and the amount can be influenced by the further production of other pro-inflammatory mediators (IL-6).15 Furthermore, IL-10 and TNF-α are cytokines that are differentially expressed by various phenotypes of macrophages, which have the opposite effect on foreign biomaterials.29 As a result, in the initial stage (12 h) of inflammatory response (in the case of 1.0−5.0 μm DW), there is a trend that TNF-α and IL-10 significantly increase with tunable responses to the DW, and IL-6 remains stable without triggering by TNF-α. In the serious stage (24 h) of inflammatory response (in the 0.22−0.65 μm sample), the anti-inflammatory response fails (IL-10 declines) and the proinflammatory response hold the great advantage of IL-6 increase. The proteolytic activity of MMPs is involved in extracellular matrix degradation, and MMPs must be precisely regulated by their endogenous protein inhibitors (the tissue inhibitors of metalloproteinases, TIMPs).30,31 In order to evaluate the mechanism of extracellular matrix degradation influenced by DW, MMP-2, MMP-9, and TIMP-1 were measured. Figure 6 shows the expression of inflammation-related proteins determined by ELISA. Figure 6a shows the expression of MMP-2. Only MMP-2 expression in the 0.22−0.65 μm sample is significantly higher than the control, reflecting that DW in the 0.22−0.65 μm range can enhance the expressions of MMP2. Figure 6b shows the expression of MMP-9. MMP-9 expression in all DW samples is significantly higher than the control. Samples of 0−0.22 and 1.0−5.0 μm have significantly lower expression than the samples of 0.22−0.65 and 0.65−1.0 μm. Figure 6c shows the expression of TIMP-1. There is no significant difference between the control and the 0−0.22 μm sample. Furthermore, the lowest TIMP-1 expression is found in 0.22−0.65 and 0.65−1.0 μm samples, but there is no significant difference between the two samples. Except for the 0−0.22 μm sample, expression of TIMP-1 increase with increasing size. 3.4. Osteoblast Cytotoxicity. Figure 7a shows the viability of osteoblasts evaluated by CCK-8 after culture with different sizes of DW for 1 and 3 d. At 1 d, the control and 0−0.22 μm sample have significant higher viability than the samples of 0.22−0.65, 0.65−1.0, and 1.0−5.0 μm DW. From 1 to 3 days, only the control and the sample of 1.0−5.0 μm exhibits significant proliferation. Figure 7b shows differentiation ability of osteoblasts assessed by measuring the ALP activity. ALP activity only in the control increase from 1 to 3 days, whereas ALP activity in DW samples decrease from 1 to 3 days. ALP activity in the control is significantly higher than that in DW samples, but no significant differences are found between DW samples. Figure 8 shows fluorescence microscope images of osteoblasts stained by rhodamine 123 (green) and DAPI (blue) after exposure to different sizes of DW after culture for 1 and 3 days. Osteoblasts after exposure to DW have smaller numbers of cells and exhibit less cell spreading. Only the control and the sample of 1.0−5.0 μm have obvious proliferation from 1 to 3 days. These data suggest that morphology of osteoblasts after exposure to DW in the range of 1.0−5.0 μm exhibit the least inhibition. In exploring the mechanisms of inflammatory and osteogenesis responses resulting from DW exposure, osteoprotegerin (OPG) and receptor of activator of nuclear factor kB-ligand (RANKL) were identified as two key factors that regulate the balance process of bone metabolism.23 According to the signal pathway analysis, RANKL promotes bone resorption by

Figure 5. (a) TNF-α, (b) IL-6, and (c) IL-10 release from cells after exposure to different sizes of DW for 12 and 24 h.

exposure to particles released more TNF-α than the control at both time points (12/24 h). Figure 5b shows the release of IL-6 after 12 and 24 h culture. There is no significant difference between particle groups at 12 h, and the 0.22−0.65 μm group has the greatest amount of IL-6 released at 24 h. Furthermore, only the 0.22−0.65 μm group has a significant increase in IL-6 release from 12 to 24 h. Figure 5c shows the release of IL-10 after 12 and 24 h culture. Macrophages after exposure to DW release more IL-10 than the control at both time points. The 0.22−0.65 μm group has the least amount of IL-10 released among the particle groups at 24 h. Furthermore, only the 0.22−0.65 μm group has a significant decrease in IL-10 release from 12 to 24 h. In summary, macrophages after exposure to DW in the 0.22−0.65 μm range release the highest amount of proinflammatory cytokines and exhibit the lowest release of antiinflammatory cytokines among all DW-induced samples. Moreover, TNF-α shows no significant increase, while IL-6 significantly increase and IL-10 decrease from 12 to 24 h in 0.22−0.65 μm DW samples. In comparison, DW of 1.0−5.0 μm significantly elevate TNF-α and IL-10, but there is no significant difference in IL-6 from 12 to 24 h. TNF-α is one of the main pro-inflammatory factors that regulates inflammaE


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Figure 7. (a) Osteoblast viability and (b) ALP activity after exposure to different sizes of DW at 1 and 3 days.

Figure 9b shows the RANKL secretion at 1 and 3 days in each group. The RANKL secretion of the control cells is significant lower than that of the DW-treated cells at 1 day. However, there is no significant difference between cells treated with different sized DW. These data suggest that RANKL secretion of DW samples is higher than that of the control. Figure 9c shows the effect of various sizes of DW on the OPG/ RANKL ratio. Except for 0−0.22 μm DW, treatment with DW from 0.22 to 2 μm resulted in an increase in OPG/RANKL ratio with increasing size. Meanwhile, only the cells treated with 0.22−0.65 and 0.65−1.0 μm DW have no significant increase in OPG/RANKL ratio from 1 to 3 days. 3.5. Fibroblast Cytotoxicity. The fibroblast viability after exposure to different sizes of particles at 1 and 3 d determined by the CCK-8 assay is shown in Figure 10. As shown, fibroblasts after exposure to DW of 0.22−0.65 μm have the smallest OD value and the control group have the largest OD value at both time points. Furthermore, only the cells treated with 0.22−0.65 μm DW exhibit inhibition of proliferation of fibroblasts from 1 to 3 d. Hence, fibroblast viability increases with increasing size of DW in the 0.22−5.0 μm range. Figure 11 shows fluorescence microscope images of fibroblasts incubated with different sizes of DW stained by rhodamine 123 (green) for 1 and 3 d. The control/0.22−0.65 μm sample groups has the largest/smallest number of cells and spreading morphology. These data suggest that fibroblasts exhibit an improvement in morphology with increasing size in the 0.22−5.0 μm range.

Figure 6. Expressions of inflammation-related proteins (a) MMP-2, (b) MMP-9, and (c) TIMP-1 after cells were exposed to different sizes of DW for 24 h.

bonding with receptor activator of nuclear factor κB (RANK), boosting osteoclast differentiation and activation. On the other hand, OPG improves bone formation by suppressing the RANKL-RANK interaction by binding to RANKL, boosting osteoblast activity and differentiation. In addition, various studies have proven that bone diseases or pathogenesis of wear debris is related to alterations in the OPG/RANKL/RANK system.32,33 Thus, the OPG/RANKL ratio may be a critical index for evaluating osteolysis. The ELISA results of OPG secretion from osteoblasts are shown in Figure 9a. At 1 day, OPG secretion of the control cells is higher than that of the experimental groups, and the cells in the 0−0.22, 0.22−0.65, and 0.65−1.0 μm groups secrete significantly less OPG than the control cells. At 3 days, only the 0.22−0.65 and 0.65−1.0 μm cells secrete significantly less OPG than the control cells. These data suggest that 1.0−5.0 μm DW has the least inhibitory impact on OPG secretion.

4. DISCUSSION During the service of artificial joint, osteolysis reactions will start from inflammatory response induced by wear debris. And then, osteoclastogenesis will be enhanced, which will be accompanied by the changing of fibrogenesis.34,35 DLC have F


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Figure 8. Fluorescence microscope images of osteoblasts stained with rhodamine 123 (green) and DAPI (blue) after exposure to different sizes of DW for 1 and 3 days.

Figure 10. Fibroblast viability after exposure to different sizes of DW at 1 and 3 days determined by the CCK-8 assay.

phages), osteoclastogenesis (osteoblasts), and fibrogenesis (fibroblasts). As we know, the production of wear debris can break the balanced mechanism of arthrosis, destroy the balance regulation of bone metabolism which is triggered by macrophages, osteoblasts or fibroblasts.37,38 Many reports have shown that size of wear debris play an important role in the exposure of osteoblastic lineage to metal or polymer wear debris, and the size of these metal or polymer wear debris is also the key factor leading to different degree of osteolysis.17,39,40 The objective of this study is to investigate the size-dependent effects of DW on cells. Actually, DW prepared in this study has the similar size with DW produced in vivo. Taeger G et al. have found that the DLC coating may be delaminated from an artificial joint after being implanted in vivo for 21months.41 DLC debris with a size about 10 μm may be produced, which can be estimated from the delamination surface10,42 of a DLC-coated joint. If the DLC coating is firmly adhered on the artificial joint and there is not any DLC delamination during its service in vitro (in PBS solution), DLC wear debris are very hard to be collected,43 because of its excellent corrosion resistance, wear resistance and the circulation of body fluid. There is not any evidence about the size of DW produced from the DLC coated artificial joint without DLC delamination in vivo or in vitro. Although DLC wear debris can not be detected and collected in vitro or in vivo, the DLC films must wear with a certain way and the wear debris can still be generated through wear processes. If there is not any DLC delamination during DLC wear in vitro or in vivo, we believe that the size of DLC wear debris may be similar to DLC wear in air. For DLC wear in air conditions, if the DLC coating is firmly adhered on the substrate and there is not any DLC delamination during DLC wear, DW is in the nano scale

Figure 9. Effect of DW size on (a) OPG secretion, (b) RANKL secretion, and (c) the OPG/RANKL ratio (* above means compared with the control).

been revealed to increase the biocompatibility of DLC coated prosthesis,36 but there are few researches on the biological response to DLC wear debris (DW) and how DW comprehensively disturb inflammatory response (macroG


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Figure 11. Fluorescence microscope images of fibroblasts incubated with different sizes of DW stained by rhodamine 123 (green) for 1 and 3 days.

Figure 12. Schematic representation of the mechanism of interaction between different sizes of DW and BSA.

0.22 and 1.0−5.0 μm showed the lower cytotoxicity. The mechanism explain is associated with the interaction between DW and BSA, the schematic diagram is shown in Figure 12. Considering that BSA is one of the major proteins in synovial fluid, BSA also can be adsorbed by diamond like carbon (DLC) film in prosthesis interface.46 Once DW is generated from DLC film surface or comes into synovial fluid during wear, it will interact with BSA preferentially. From the results of zetapotential analysis, DW in the size range of 0−0.22 and 1.0−5.0 μm has a positive potential (Table 2). As BSA has a negative potential in physiological conditions, DW in the size range of 0−0.22 μm and 1.0−5.0 μm will attract BSA through electrostatic adsorption. DW in the size range of 0.22−0.65 and 0.65−1.0 μm has a negative potential, it will reject BSA through electrostatic repulsion. As BSA enhanced surface affinity to cells, it also could improve biocompatibility of particles.47,48 For DW in the size range of 0−0.22 and 1.0−5.0 μm, the effect of BSA-encapsulated will exhibit less cytotoxicity. Therefore, DW samples of 0−0.22 and 1.0−5.0 μm show the lower cytotoxicity than that of DW samples of 0.22−0.65 and 0.65−1.0 μm. Our results revealed that the interactions between cells and various sizes of DW lead to the differently comprehensive response of macrophages, osteoblasts and fibroblasts. Interface tissue exposed to DW has shown an increased osteoclastic area, sizes as a vital factor can contribute to varying degrees of periprosthetic osteolysis. According to joint interface related cells responded to biomaterials, the size-dependent cytotoxicity evaluation of DW provide an attractive perspective for the biocompatibility evaluation of DLC film, and thereby made the mechanism elucidation in this research represent an interesting new area of biomaterials-related research. In conclusion, the DW results of this paper show for the first time about the comprehensive regulation mechanism of macrophages, osteoblasts, and fibroblasts responding to different sizes of DW. It can be seen DW within the size range of 0.22−0.65 μm might have a significant effect on pro-

(about 1.0−5.0 nm diameter) after wear.44 Considering the DLC delamination and the DLC wear without delamination in vivo, the size of the DW generated in vivo may be ranged from 50 nm to 10 μm. In our research, the size range of DW was 0− 5.0 μm, which was similar to DW produced in vivo. DW in the size ranges of 0−0.22, 0.22−0.65, 0.65−1.0, and 1.0−5.0 μm was chosen and prepared, and their size-dependent cellular response was evaluated in this research. Through the cell activity researches of macrophages, osteoblasts and fibroblasts, we found that exposure of cells (related to inflammatory response, osteoblastogenesis, and fibrous reaction) to DW (within a certain size range) resulted in related cells in a osteoclastogenic trend, expressing more TNF-α, IL-6, MMP-2, MMP-9, and RANKL (shown in Figures 5a, 5b, 6a, 6b, and 9b). When Raw 264.7 macrophage cells exposed to DW of different sizes, the expression of the key factors related to pro-inflammatory factors and MMP family factors (MMP-2 and MMP-9) was up-regulated, which led the way to the expression of osteoclast signal factor (RANKL). RANKL was the product of osteoclastic response, and its expression also continuously enhanced the osteoclastogenic trend through the other molecular mechanism.45 Compared with the control, the results reflected that DW induced osteoclastogenesis, and osteoclast activities were also significantly enhanced continuous interaction with DW. Once biological tissue is exposed to DW, macrophages mediated activities immediately came up and related inflammatory factors release also triggered a series of osteoclastogenesis. We found that except the DW with size range of 0−0.22 μm, DW cytotoxicity showed a size-dependent (0.22−5.0 μm) decrease with increasing size. The DW sample of the size range of 0.22− 0.65 μm had the highest osteoclastogenesis (shown in Figures 7, 8, and 9). Larger sizes of DW had less stimulatory effect on the cytomembrane site and less chance of inducing phagocytosis. The results also showed that comparing with the DW samples of 0.22−0.65 and 0.65−1.0 μm, DW samples of 0− H


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inflammatory and pro-osteoclastic cytokine release. Thus, the cytotoxicity research of different sizes of DW enrich the knowledge of biocompatibility evaluation of DLC-coated prosthesis, and the findings of this paper will greatly promote the application of DLC modification.

5. CONCLUSIONS In this study, various size ranges (0−0.22, 0.22−0.65, 0.65−1.0, and 1.0−5.0 μm) of DW was successfully generated and isolated. Except for the size range of 0−0.22 μm, DW cytotoxicity showed a size-dependent (0.22−5.0 μm) decrease with increasing size. DW of larger size had a less negative effect on cell adhesion and growth, resulting in less inflammatory reaction and excellent osteogenic and fibroblastic responses.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86 28 87601149. Fax: +86 28 87601149. ORCID

T. T. Liao: 0000-0002-7005-0252 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (31570958), Science and Technology Support Program of Sichuan Province (2016SZ0007), and the Fundamental Research Funds for Central Universities (2682016YXZT07).

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REFERENCES

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Evaluation of the Size-Dependent Cytotoxicity of DLC - American

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