Insight into the wear particles of PEEK and CFRPEEK against UHMWPE for artificial cervical disc application: Morphology and immunoreaction

Journal Pre-proof Insight into the wear particles of PEEK and CFRPEEK against UHMWPE for artificial cervical disc application: Morphology and immunoreaction Jian Song, Fangfei Chen, Yuhong Liu, Song Wang, Xiang He, Zhenhua Liao, Xiaohong Mu, Mengying Yang, Weiqiang Liu, Zhongxiao Peng PII:

S0301-679X(19)30607-3

DOI:

https://doi.org/10.1016/j.triboint.2019.106093

Reference:

JTRI 106093

To appear in:

Tribology International

Received Date: 24 September 2019 Revised Date:

22 October 2019

Accepted Date: 28 November 2019

Please cite this article as: Song J, Chen F, Liu Y, Wang S, He X, Liao Z, Mu X, Yang M, Liu W, Peng Z, Insight into the wear particles of PEEK and CFRPEEK against UHMWPE for artificial cervical disc application: Morphology and immunoreaction, Tribology International (2020), doi: https://doi.org/10.1016/ j.triboint.2019.106093. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Insight into the wear particles of PEEK and CFRPEEK against UHMWPE for artificial cervical disc application: morphology and immunoreaction

Jian Song a, 1, Fangfei Chen b, 1, Yuhong Liu a, *, Song Wang c, Xiang He d, Zhenhua Liao c, Xiaohong Mu e, *, Mengying Yang a, Weiqiang Liu a, c, *, Zhongxiao Peng f

a.

State Key Laboratory of Tribology, Tsinghua University, Beijing 100084, China.

b.

Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China.

c.

Key Laboratory of Biomedical Materials and Implant Devices, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057, China.

d.

State Key Laboratory of Powder Metallurgy, Central South University, Changsha 410083, China.

e.

Department of Osteology, Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, Beijing 100700, China.

f.

School of Mechanical and Manufacturing Engineering, The University of New South Wales, Sydney 2052, Australia.

*

Corresponding authors:

Y. Liu (Tel: +86-10-62788387, email: [email protected]) X. Mu (Tel: +86-10-84013324, email: [email protected]) W. Liu (Tel: +86-755-26551376, email: [email protected]). Present address: J. Song (Department of Mechanical Engineering and Munich School of Bioengineering, Technical University of Munich, Garching 85748, Germany)

1

Abstract： ：Artificial cervical disc is a key device for artificial cervical disc replacement (ACDR). In our previous study, we developed PEEK-on-UHMWPE and CFRPEEK-on-UHMWPE tribo-parings for artificial cervical disc application. To further evaluate the biotribological properties and potential inflammation responses of their by-products, the size distribution and morphological characteristics of collected post-test wear particles as well as the biological response of aggregated particles were studied. Our results indicated that the majority of these wear particles were in a size range of 0.05-25 µm and the possibility of immunoreaction was small. Thus, we provide further evidence from the wear particles' point of view to suggest that CFRPEEK-on-UHMWPE can be considered as the potential paring for artificial cervical disc application.

Keywords: wear particle; CFRPEEK; morphology; immunoreaction

2

1 Introduction Spinal pain, including low back and neck pain, is becoming a large and rapidly increasing health topic and challenging health-care systems worldwide

1

because associated diseases can

result in patients’ disability. One of the main reasons might be attributed to the degenerative cervical conditions, also expressed as degenerative disc disease (DDD) 2. As an alternative to anterior cervical discectomy and fusion (ACDF), artificial cervical disc replacement (ACDR) surgery, which can relieve pain and restore the physiological motion function of the spinal segment, has been served as the next frontier in surgical treatment for DDD. Normally, the shape of the bearing surfaces of artificial cervical discs for DDD is designed to be ball-on-disc. More specifically, these bearing combinations can be roughly divided into three groups: metal-on-metal (MoM), metal-on-polymer (MoP) and polymer-on-polymer (PoP). The MoM and MoP are the conventional ones and widely used in clinical applications 3. It was reported that the MoM and MoP bearings may undergo ion toxicity and severe wear damage after mid- and long-time implantation

4, 5

. One possible solution is to develop PoP bearing with

excellent wear resistance and biomechanical properties. Recently, poly-ether-ether-ketone (PEEK) and its composites are widely investigated owing to their great potential to be the bearing materials of new generation PoP artificial cervical disc 6-8. It is noted that the PEEK PoP artificial cervical discs are mainly PEEK self-mating bearing couples

9, 10

. Thus, we developed novel

bearing combinations of PEEK-on-ultra-high molecular weight polyethylene (UHMWPE) and CFRPEEK-on-UHMWPE for artificial cervical disc application 11. The long-term wear resistance and wear mechanisms of the two bearings were studied. The results indicated that they offer better tribological properties in comparison with the conventional ones. It has been reported that wear particles generated from the prosthetic joint articular surface may lead to aseptic loosening and further cause the failure of implantation 12. Thus, evaluation of the tribological properties and biological reactions of wear particles of artificial joints is of importance. Normally, the morphological properties of wear particles of artificial joints were investigated using scanning electron microscopy

13-15

and the morphological parameters, e.g.,

equivalent circular diameter (ECD), roundness (R), aspect ratio (AR), form factor (FF) and elongation factor (EF), were studied based on the obtained images with ASTM F1877 standard 16. Gladkis et al.

17

also evaluated the size, shape and abundance of UHMWPE wear particles from

knee prostheses using atomic force microscopy, which can be used as an alternative to filtration 3

and SEM. Moreover, there is a direct relationship between the presence of osteoblasts and macrophages in the tissue and wear particles

18, 19

. Thus, the in vitro and in vivo osteoblasts and

macrophages responses to wear particles of artificial joints are widely investigated 20, 21. However, the properties of wear particles are decided by experimental conditions and bearing combinations. Although the above studies give good examples to investigate the wear particles of artificial joints, further wear particles studies are required for PEEK-on-UHMWPE and CFRPEEK-on-UHMWPE, which are novel bearing combinations for artificial disc application 11. The work presented here is motivated by such considerations. The wear particles of PEEK-on-UHMWPE and CFRPEEK-on-UHMWPE were isolated from the lubricants after the long-term in vitro wear simulation using the enzymatic digestion method. The size distribution and morphological properties were studied. Moreover, we describe how different amount, i.e., the degree of particle aggregation, can affect the biological response of PEEK and CFRPEEK particles.

A

better

understanding

of

the

potential

of

PEEK-on-UHMWPE

and

CFRPEEK-on-UHMWPE bearing combinations for artificial disc application will be achieved from the wear particles' point of view.

4

2 Materials and methods 2.1 Wear particle generation and isolation The long-term wear evaluation of PEEK-on-UHMWPE and CFRPEEK-on-UHMWPE were conducted in

11

using an in vitro wear simulator. In brief, the tests were carried out for 5 MC

(million cycles) in a frequency of 1 Hz based on ISO 18192-1 standard. Newborn calf serum (Hangzhou Si Ji Qing Co., Ltd, China) with a protein concentration of 20 g/L was utilized as a lubricant. For each bearing combination, the lubricant after every 0.5 or 1 MC was collected and fresh calf serum was used. Unlike the lubricants utilized for industrial application, simulated body fluids, such as calf serum, are chosen as lubricants to simulate the body conditions. As a result, the macromolecules, such as proteins, often adhere to wear particles generated from the discs and influence further analysis. Therefore, filtration and isolation of the wear particles from the post-test lubricants are necessary. There are three methods for digesting the proteins and isolating the wear particles for the post-test lubricants: enzymatic digestion 22, 23, alkali digestion 24, 25, and acid digestion 26, 27. In terms of the enzymatic digestion, some enzymes, such as hyaluronidase, trypsin, and proteinase K, are applied to digest the macromolecules in the lubricants, and then the wear particles can be obtained after filtration. This method is relatively easy with little waste. The wear particles evaluated in this study were isolated from the collected calf serum samples according to the enzymatic digestion protocol described in 22, 23. The calf serum with wear particles was first prepared by ultrasonic dispersion for 30 min. Next, 5 mg/mL hyaluronidase (H3506, Sigma, St. Louis, MO), 2 mg/mL trypsin (T4799, Sigma, St. Louis, MO) and 1.8 mg/mL proteinase K (V900887, Sigma, St. Louis, MO) were dissolved with pure water. Then, the dispersed lubricant and enzyme solution were mixed in a ratio of 1:1 (v/v) at 37 ℃. After interaction for 12 h, the mixture was diluted with pure water to a ratio of 10:1 and dispersed by ultrasonic for 30 min again. Finally, the whole solution was filtered through 50 nm filter (Whatman, Kent, UK), and the cleaned wear particles were obtained with the filter (see Fig. S1).

2.2 Wear particles characterization Since wear particles are closely related to the wear modes and mechanisms 28, studying the

5

morphological properties of wear particles can assist in further evaluation of the tribological characteristics of the tribo-pairs. Particle size distributions of the post-digestion lubricants were evaluated by a dynamic light scattering technique (DLS) using a commercial zetasizer (Malvern Nano-ZS, Worcestershire, UK). Then, the isolated wear particles were studied using scanning electron microscopy (SEM, FEI Quanta 200FEG, Eindhoven, The Netherlands). In order to improve the image quality, the particle samples were coated with a thin platinum (Pt) layer before SEM observation. For each bearing combination, images of over 100 wear particles with different sizes and morphological features were captured. Then, the morphological properties were analyzed using professional software “ImageProPlus” (v.6.0.0, Media Cybernetics，Silver Spring， USA) and according to the ASTM F1877 standard. As displayed in Table 1, five numerical parameters, including equivalent circular diameter (ECD), roundness (R), aspect ratio (AR), form factor (FF) and elongation factor (EF), were used to measure the shape features of the wear particles in this study. Table 1 The morphological parameters for wear particle characterization. No.

Parameter

1

ECD

2

R

3

AR

4

FF

5

EF

Definition (

4

Meaning A measure of size

)

4A

A measure of how closely the particle resembles a circle (a perfect circle having a value of one) A common measure of shape

4

A measure of how closely the particle resembles a circle based on perimeter A measure of shape, especially for a much longer particle

A: particle area; dmax: the longest straight line drawn between any two points on the particle; dmin: the longest line perpendicular to the major diameter; p: particle perimeter; FL: particle length; FW: particle breadth.

2.3 Biological assays The aggregation of wear particles can lead to aseptic loosening by stimulating cell reactions (e.g., cell activation, cytokine release and hypersensitive reaction)

29

. In order to explore the

influence of the aggregation of wear particles on the human body, cellular morphology observations and immunoreaction tests of PEEK and CFRPEEK particles were carried out according to ISO 10993-5 standard. Mouse osteoblast-like cells (MC3T3-E1, Procell, Wuhan, China) and mouse macrophages (ANA-1, Procell, Wuhan, China) were purchased and grown in a 6

culture fluid of RPMI 1640 medium 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin solution according to the manual. Cells were incubated at 37 ℃ in a 5% CO2 incubator and the medium was replaced every 48 h until the cell suspension with a count of 5×104/mL was obtained. Owing to the excellent wear resistance of PEEK and CFRPEEK 11, it is difficult to generate and obtain a large number of wear particles. Also, separation of PEEK/CFRPEEK and UHMWPE particles is very challenging if possible. Thus, to investigate the biological reactions of PEEK and CFRPEEK particles individually, particles of PEEK (450G, Victrex, UK) and CFRPEEK (450CA30, Victrex, UK) were manually prepared using a commercial ultrafine grinder. According to the user manual, the knife blades were made of steel (X46Cr13, 1.4304) with a hardness of 245 HB, which is much harder than the PEEK and CFRPEEK. Thus, we assumed that the vast majority of the particles obtained were PEEK or CFRPEEK ones. These particles were then examined with an SEM (FEI Quanta 200FEG) to ensure similar morphologies and sizes with the wear particles obtained from the in vitro tests in Section 2.2 (see Fig. S2). Both PEEK and CFRPEEK particles were divided into four groups with different mass (0.1 mg, 0.5 mg, 1.0 mg, and 5.0 mg), and then they were autoclaved for 30 min for further use. PEEK and CFRPEEK particles with four different mass values mass (0.1 mg, 0.5 mg, 1.0 mg, and 5.0 mg) were ultrasonically dispersed in 1 mL culture fluid, respectively. The control groups involved the use of 0.64% phenol culture fluid as positive controls and culture fluid without any particles as negative controls. Then, all kinds of the sample fluids were added into the corresponding wells of the 24-well plate containing the mouse osteoblastic cells and mouse macrophages cell suspensions, respectively. Then, all the wells were filled with medium. Cells were incubated at 37 ℃ in a 5% CO2 incubator. The shape and aggregation of cells with different particles were examined under an inverted microscope (Motic AE30) after 24 h. The IL-1β and TNF-α cytokines of the cell culture supernatants were detected with enzyme-linked immunosorbent assay (ELISA) with a culture time of 48 h according to the manufacturer's instructions (DKW12-2720, Dakewei, Shenzhen, China).

2.4 Statistics In this study, the obtained results were statistically evaluated by one-way ANOVA and least-significant difference (LSD) post hoc tests by using a professional software “SPSS” (v20.0.0,

7

SPSS Inc., Chicago, USA). The p-values less than 0.05 were considered to be statistically significant.

3 Results and discussion 3.1 Wear particles characterization Separation of PEEK/CFRPEEK and UHMWPE particles is very challenging owing to their similar properties and our technical limitation. Thus, we just study the wear particles generated by different material combinations, which still make sense because both of the bearing materials contribute to the wear particles production from the tribological point of view. As shown in Fig. 1, the size distributions of the wear particles of PEEK-on-UHMWPE and CFRPEEK-on-UHMWPE artificial discs. The data reveals that the vast majority of the wear particles of the PEEK-on-UHMWPE and CFRPEEK-on-UHMWPE artificial discs is in a size range from 0.05 µm (pore size of filter) to 25 µm. It was suggested that the larger wear particles of artificial joints were observed by SEM compared to TEM, which were thought to originate from the wear stripe 30. The size values of the wear particles presented in this work are relatively higher than the ones shown in a previous study

31

, which reported the wear particles generated by PEEK-ceramic

coupling for artificial disc application demonstrated a mean equivalent circular diameter value ranging from 0.1 µm to 16.69 µm. Fig. 1 also illustrates that the wear particles of PEEK-on-UHMWPE

paring

are

relatively

larger

than

the

ones

generated

by

CFRPEEK-on-UHMWPE, which are in accordance with the results obtained in previous fretting wear tests

32

. This may attribute to the carbon fibers in CFRPEEK, which can carry the load

during frictional motion and restrict the removal of materials in the composite matrix. As a result, smaller

wear

particles

of

CFRPEEK-on-UHMWPE

PEEK-on-UHMWPE.

8

are

observed

compared

to

Fig. 1 Size distributions of the wear particles obtained from the in vitro wear tests by a dynamic light scattering technique (DLS).

However, size distribution is just a qualitative analysis of the wear particles. In a next step, the morphological properties of those wear particles were quantitatively characterized and the mean values plus standard deviation are presented in Table 2. The results indicated that the wear particles of the two artificial discs had a large standard deviation of ECD and AR, suggesting a broad range of size and various shape of the particles. Compared with PEEK-on-UHMWPE, the wear particles of CFRPEEK-on-UHMWPE are smaller with a relative lower ECD, which is in agreement with the results shown in Fig. 1. According to the statistical analysis of the five morphology related parameters shown in Table 2, there is no significant difference between PEEK-on-UHMWPE and CFRPEEK-on-UHMWPE artificial discs (p > 0.05), indicating the mean size and shape features of the wear particles from the two pairs were also similar. Thus, for the following wear mechanisms analysis, we just focused on the other wear mechanism related features of the particles without distinguishing the bearing combinations. One should mention here is that the ECDs of the wear particles obtained from the PEEK/CFRPEEK-on-UHMWPE bearings are higher than the ones from metal-on-metal (typical size: 50 nm) and metal-on-UHMWPE (typical size: 0.5 µm)

33

. Interestingly, we can find that the magnitude of the size of

metal-on-metal, metal-on-UHMWPE and PEEK/CFRPEEK-on-UHMWPE increases one by one. We attribute it to the different hardness and materials properties of metallic and polymeric materials.

We

also

would

like

to

remind

that

the

in

vitro

wear

tests

of

PEEK/CFRPEEK-on-UHMWPE artificial disc prostheses were conducted in a modified knee simulator, which may also cause some errors for the wear particles. In addition, the numbers of small particles cannot against the contribution made by large ones, leading to a large average size

9

with high standard deviation. Table 2 Morphological characterization of the wear particles of artificial discs. The error values shown depict the standard deviation. PEEK-on-UHMWPE

CFRPEEK-on-UHMWPE

Equivalent circular diameter (ECD, µm)

13.3 ± 9.1

11.4 ± 7.2

Aspect ratio (AR)

1.90 ± 0.96

1.92 ± 0.98

Roundness (R)

0.44 ± 0.16

0.46 ± 0.15

Form factor (FF)

0.38 ± 0.14

0.40 ± 0.16

Elongation factor (EF)

0.37 ± 0.21

0.41 ± 0.20

As displayed in Fig. 2, there are five kinds of representative wear particles generated from the in vitro wear tests. More specifically, the flake-like wear particles samples in Fig. 2(a1-3) have irregular flat shapes, which are the common particles observed in this study and can be roughly divided into three groups. Fig. 2(a1) shows a representative particle with a large size and some plowing grooves on the surface, indicating that the particle was squeezed and abraded during their generation process. As can be found in Fig. 2(a2), this kind of flake-like particles usually have a size of 10-30 µm and stereoscopical curling edges. This might be attributed to fatigue cracks caused by the motion of articulation. Owing to the specific structure of artificial disc prosthesis, stress would be concentrated within the small contact area at the interface between the UHMWPE inlay and PEEK/CFRPEEK end plates 34, which leads to fatigue wear and then generates fatigue cracks. The cracks usually expand with the increasing motion cycles and can be removed from the bearing surfaces with adhesion wear. As a result, some fatigue cracks are found on the PEEK/CFRPEEK concave bearing surfaces in our previous work 11. The third type of flake-like particles in Fig. 2(a3) is relatively smooth and small in size, which is associated with fatigue wear mechanism. However, we here just provide a brief explanation of the wear mechanism from the wear particles' point of view. Further comparison of the surface roughness values and particle sizes are needed to better understand the wear mechanisms.

10

Fig. 2 Representative SEM images of wear particles obtained from the in vitro wear tests: (a1-3) flake-like wear particles, (b1-3) spherical wear particles, (c1-3) aggregated wear particles, (d1-3) rod-like wear particles, and (e1-3) zonal wear particles.

Spherical wear particles, also common particles obtained in the wear tests, are presented in Fig. 2(b1-3). Normally, the spherical particles are small with ECD < 5 µm. It is suggested that smaller particles are usually generated from the fragmentation of larger ones or the exfoliation of surface micro-convex-bodies of friction, resulting in a spherical shape with the lowest energy status

15

. Therefore, these spherical particles are contributed by adhesion wear or the 11

fragmentation of large wear particles. In addition, owing to their small size, these spherical particles can aggregate easily. Regarding the aggregated particles displayed in Fig. 2(c1-3), they have irregular outlines and are in a wide size range from a few microns to tens of microns. The particles are aggregated and can further adhere to each other under the interaction of several mechanisms to create a larger aggregated object

35

. In addition to the spherical ones, small particles in other shapes are also

found in the aggregation. As shown in Fig. 2(d1-3), rod-like wear particles were also obtained in this study. They are formed by rubbing the peeled materials of the bearings continuously, reflecting the motion combination of artificial discs in the wear tests, which is similar to the rubbing wear particles of engine oil 36. It is noted that the length of the rod particle is much larger than its width, which can further covert to other types. Thus, the number of rod-like particles is lower in comparison with the above ones. Fig. 2(e1-3) demonstrate the typical morphology of zonal wear particles. Compared with the rod wear particles, the zonal particles are shorter. However, their lengths are still twice of the widths. As can be seen, zonal particles display smoother surfaces and clear edges. Here we assume their formation mechanism, similar to the rod-like particles, is dominated by peeling and fragmentation after fatigue wear. In a comparable study 14, polymeric particles of artificial disc with a shape of sphere, fibril, and flake were reported. Compared with artificial hip or knee joints, the wear particles generated by artificial discs are smaller and more round

37

. It has been proposed that the wear particles

within the size range of 0.1-10 µm are the most reactive for the immunoreaction of macrophage, which can further lead to the aseptic loosening of the implanted artificial disc

18

. Therefore,

biological tests of these particles were conducted and the results can be found in Sections 3.2 & 3.3.

3.2 Cellular morphology To date, the biological response of UHMWPE wear particles generated by artificial joints and artificial discs has been widely investigated

38, 39

. Therefore, only the cellular morphology and

immunoreaction assays of PEEK and CFRPEEK particles were conducted in this study. Due to our technical limitation, it is hard to isolate the manually obtained wear particles into different 12

groups according to their different morphologies. Thus, here we just used different mass values to simply mimic the accumulation of wear particle after different service time. Fig. 3 displays the optical micrographs of osteoblastic cells incubated with different mass values of PEEK particles at 37 ℃ for 24 h, 48 h and 96 h. As can be seen in Fig. 3(a), the normal osteoblasts in negative control group are polygonal or fusiform cells with a single nucleus. With increasing PEEK particles, the proliferation of osteoblasts is inhibited, as indicated by the drastically decreased cells, suggesting osteoblasts are sensitive to the amount of PEEK particles. When the amount of PEEK particles reached 1.0 mg and above, the osteoblasts changed into a much shorter one, and some of them gathered around the particles, demonstrating signs of phagocytosis, as shown in Fig. 3(d-e). Similar osteoblasts phagocytosis of titanium and CoCr wear particles have been also reported in a previous study 19, revealing phagocytosed particles can further lead to the morphological changes on the cells. After incubating with PEEK particles for 96 h, there were still some osteoblasts survived, indicating less toxicity compared to phenol in the positive control, where nearly all cells went through apoptosis (see Fig. 3(f)).

13

Fig. 3 Optical micrographs of osteoblasts incubated with different numbers of PEEK particles at 37 ℃ for 24 h (left), 48 h (middle) and 96 h (right). The control groups involved the use of 0.64% phenol culture fluid as positive controls and culture fluid without any particles as negative controls. All images are obtained at 200x magnification.

The cellular morphologies of osteoblasts incubated with different amounts of CFRPEEK particles and durations are shown in Fig. 4. With the increasing CFRPEEK particles, a similar but less obvious declining trend of cell density was observed in comparison with the results of PEEK displayed in Fig. 3. The results indicated that CFRPEEK can inhibit the proliferation of osteoblasts as well, but not as much as PEEK. As shown in Fig. 4(d-e), the cellular morphology changed with different amounts of CFRPEEK particles. After incubated with CERPEEK particles for 96 h, the shape of osteoblasts turned into strip-type from polygonal or fusiform and arranged

14

sparsely, indicating disturbance to their normal function. It can be assumed that the CFRPEEK particles tend to induce elongation of the osteoblasts while PEEK particles make them adapt shorter appearances.

15

Fig. 4 Optical micrographs of osteoblastic cells incubated with different numbers of CFRPEEK particles at 37 ℃ for 24 h (left), 48 h (middle) and 96 h (right). The control groups involved the use of 0.64% phenol culture fluid as positive controls and culture fluid without any particles as negative controls. All images are obtained at 200x magnification.

Unlike osteoblasts, wear particles mainly act through stimulating macrophage secretion of cytokines, which induces immune response and phagocytosis. Thus, only the micrographs of macrophages after incubating with PEEK and CFRPEEK particles for 24 h were observed in this study. The normal morphology of macrophages is oval or round-shaped, with abundant cytoplasm containing many granules and vesicles, as shown in Fig. 5(a). Compared to the negative control, the shape of macrophages in the positive control shown in Fig. 5(b) demonstrate an insignificant change. When particles appeared in the culture, macrophages were activated to aggregate around the particles, swallowing them by phagocytosis. With the increasing particle numbers, the cytoplasm of macrophages was filled with particles, as shown in Fig. 5(f) and (j). Those particles and in particular the ones with a large size were encompassed to form fibrous membrane, which can lead to the bone resorption

40

. Because the PEEK and CFRPEEK particles were not

decomposable, they would exist in the macrophages for a long time, stimulating the generation of various cytokines, which in turn activates immune response as well as the differentiation of osteoclasts. As a result, the dynamic balance of bone remodeling was disturbed, leading to osteolysis or disorder in bone formation around the prosthesis, and further resulting in the aseptic loosening of cervical disc prosthesis.

16

Fig. 5 Optical micrographs of macrophages incubated with different numbers of PEEK and CFRPEEK particles at 37 ℃ for 24 h. The control groups involved the use of 0.64% phenol culture fluid as positive controls and culture fluid without any particles as negative controls. All images are obtained at 400x magnification.

3.3 Immunoreaction It has been proposed that interleukin (IL)-1β and tumor necrosis factor (TNF)-α are two major cytokines that decrease bone mineral density by inhibiting osteoblast differentiation and bone formation, leading to bone loss in many inflammatory diseases

41

. Thus, IL-1 and TNF-α

cytokines of osteoblasts and macrophages with different particle amounts were evaluated with ELISA approach in this study, which can offer quick and accurate results with high sensitive 42. As shown in Fig. 6(a), IL-1β concentrations in osteoblasts of all the groups are similar, which are in a range of 60.3-60.6 pg/mL. More specifically, IL-1β concentrations in osteoblasts of all the sample groups are lower than that of the negative control. According to the results obtained from statistical analysis, there is a significant difference between 1.0 mg PEEK particles and the negative control (i.e., using culture fluid without any particles), indicating that the particles inhibited the secretion of IL-1β in osteoblasts. Regarding the CFRPEEK particles, the ones with a mass value less than 1.0 mg are all significantly different from the negative control. Thus, from the osteoblasts IL-1β secretion point of view, CFRPEEK particles demonstrate better than PEEK. Regarding the macrophages, as displayed in Fig. 6(b), IL-1β concentrations in osteoblasts of all the sample groups are also lower than that of the negative control. Meanwhile, no significant 17

differences are found between the negative control and sample groups with statistical analysis. IL-1β concentration is a key indicator of the degree of inflammation in the human body. It worth noting that the IL-1β concentrations in the two cells incubated with PEEK and CFRPEEK particles are all lower than the positive control incubated with 0.64% phenol, suggesting that the degree of inflammation caused by these particles is less than the one caused by phenol, which are in accordance with the results of cellular morphology shown in Fig. 3 and Fig. 4. It is indicated that titanium (Ti, one of the conventional materials for artificial discs) particles can induce IL-1β secretion through activation of the NALP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome complex 43. Based on the results in this study, one can conclude that it is unlikely that the wear particles generated by PEEK and CFRPEEK as cervical disc materials promote the secretion of IL-1β in osteoblasts and macrophages, suggesting better biocompatibility of PEEK and CFRPEEK in comparison with the conventional orthopedic implant materials.

18

Fig. 6 PEEK and CFRPEEK particles induced IL-1β production in osteoblasts (a) and macrophages (b) after incubating for 48 h. PEEK and CFRPEEK particles induced TNF-α production in osteoblasts (c) and macrophages (d) after incubating for 48 h. The control groups involved the use of 0.64% phenol culture fluid as positive controls and culture fluid without any particles as negative controls. Data represented mean ± SD (n= 4). *p < 0.05, negative control vs. samples & positive control.

TNF-α is the most effective proinflammatory cytokine that can activate osteoblasts to secrete IL-6 and suppress type I collagen synthesis and proliferation, which further contributes to bone resorption and leads to bone destruction and implant loosening

44

. Fig. 6(c-d) also presents the

PEEK and CFRPEEK particles induced TNF-α production in osteoblasts and macrophages after incubating for 48 h. Different from the IL-1β results shown in Fig. 6(a-b), TNF-α secretion in osteoblasts and macrophages are more sensitive to PEEK and CFRPEEK particles. As can be found in Fig. 6(c), TNF-α production volumes in osteoblasts induced by the sample groups are higher than the negative control. More specifically, when osteoblasts interacted with 0.1 mg, 0.5 mg and 5.0 mg PEEK particles, the TNF-α production volumes are statistically different from the negative control, suggesting higher secretion of TNF-α. Regarding the positive control, the introduced phenol leads to the change of cellular morphology and further lead to the inhabitation of proliferation.

Consequently, lower TNF-α concentration is found in the positive control

compared to the negative control. As shown in Fig. 6(d), for the macrophages, the TNF-α concentrations of the sample groups are all higher and that of positive control is lower compared to the negative control, which are similar to the osteoblasts. According to the results of statistical analysis, the TNF-α production volumes of sample groups are all significantly different from the negative control. Macrophage, a kind of white cells in human body, can surround the introduced particles and further phagocytize some of them (Fig. 5), simulating the inflammation response and leading to higher TNF-α production. Indeed, wear particles, as foreign substances, will influence the normal physiological function of human body and can eventually induce immunoreaction. According to the results obtained in this study, the degrees of immunoreaction of PEEK and CFRPEEK particles with the tested mass concentrations are similar. It is worth noting that the cell culture was conducted with 24-well plates in this study, indicating particle concentrations from 0.1 mg/mL to 5.0 mg/mL. Based on the results obtained from the in vitro wear tests 11, the wear rates of the two artificial discs are 3.82 mg/MC and 2.34 mg/MC, resulting in a lower particle concentration than the highest concentration of 5.0 mg/mL used in this study. Thus, it can be assumed that the possibility of immunoreaction and aseptic loosening caused by PEEK and CFRPEEK particles is small. 19

According to previous studies 45-48, it is suggested that the PEEK and CFRPEEK are non-cytotoxic, while some studies also propose that CFRPEEK is cytotoxic and cannot be used for implant material. Therefore, long-term biological assays and animal experiments should be carried out to further evaluate the PEEK wear particles’ and CFRPEEK wear particles’ physiological significance and roles in aseptic loosening.

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4 Conclusions In this presented work, we have evaluated the morphological properties and immunoreaction of the wear particles of PEEK-on-UHMWPE and CFRPEEK-on-UHMWPE artificial cervical discs that we developed in a previous study 11. The size distribution, morphological properties and wear mechanisms of the wear particles isolated from lubricants in the long-term in vitro wear simulation were analyzed. It has been found that most of the wear particles of the two artificial discs are in a range of 0.05-25 µm. Five kinds of representative wear particles have been captured: flake wear particles, spherical wear particles, aggregated wear particles, rod wear particles, and zonal wear particles. We also have conducted the cellular morphology observation and immunoreaction assays of different mass values of PEEK and CFRPEEK particles to explore the influence of the aggregation of wear particles on the human body. Regarding the equivalent particles concentration in the in vitro wear tests, the possibility of immunoreaction and aseptic loosening caused by PEEK and CFRPEEK particles from the cervical disc prosthesis is small. Certainly, both PEEK and CFRPEEK wear particles will come with their own, detailed challenges when taking real human body condition into consideration. The data presented here helps better understand the related wear mechanisms of the wear particles and motivates that PEEK-on-UHMWPE and CFRPEEK-on-UHMWPE can indeed serve as powerful bearing combinations in artificial cervical disc application for further studies.

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Acknowledgments The authors appreciate Dr. Jing Yang, from China Academy of Chinese Medical Sciences, for expert assistance with biological assays. This project has received funding from the National Key R&D Program of China (Grant No. 2016YFC1101803), the National Natural Science Foundation of China (Grant No. 51875303), the Program for New Century Excellent Talents in University (Grant No. NCET-12-0805) and the International Science & Technology Cooperation Program of Shenzhen (Grant No. GJHZ20180413181811215).

Conflicts of interest The authors declare no conflict of interest.

Author Contributions J. Song and F. Chen contributed equally to this work. J. Song, F. Chen and X. Mu proposed the study. J. Song conducted the experiments. Y. Liu, W. Liu and X.Mu provided the funding and material support as well as study supervision. The manuscript was written by J. Song and F. Chen. All authors contributed to the analysis and discussion of the data, reviewed the manuscript, and gave approval to the final version of the manuscript.

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High lights: The wear particles were in a size range of 0.05-25 µm. The possibility of immunoreaction induced by the particles was small. CFRPEEK-on-UHMWPE can be considered as a paring for artificial cervical disc.

State Key Laboratory of Tribology Tsinghua University Beijing 100084, China E-mail: [email protected] Tel: 86-10- 62788387 October 22, 2019

Dear Prof. Dr. Michel Fillon, As the corresponding author, I hereby declare, on behalf of all authors, no competing financial interest. Thank you and best regards. Yours sincerely,

刘宇宏 Yuhong Liu