# Abstract

 Cervical degenerative disease is one of the most common spinal disorders worldwide, especially in older people. Anterior cervical corpectomy and fusion (ACCF) is a useful method for the surgical treatment of multi-level cervical degenerative disease. Anterior cervical disc replacement (ACDR) is considered as an alternative surgical method. However, both methods have drawbacks, particularly the neck motion decrease observed after arthrodesis, and arthroplasty should only be performed on patients presenting with cervical disc disease but without any vertebral body disease. Therefore, we designed a non-fusion cervical joint system, namely an artificial cervical vertebra and intervertebral complex (ACVC), to provide a novel treatment for multi-level cervical degenerative disease. To enhance the long-term stability of ACVC, we applied a hydroxyapatite (HA) biocoating on the surface of the artificial joint. Thirty-two goats were randomly divided into four groups: a sham control group, an ACVC group, an ACVC-HA group, and an ACCF group (titanium and plate fixation group). We performed the prosthesis implantation in our previously established goat model. We compared the clinical, radiological, biomechanical, and histological outcomes among these four different groups for 24 weeks post surgery. The goats successfully tolerated the entire experimental procedure. The kinematics data for the ACVC and ACVC-HA groups were similar. The range of motion (ROM) in adjacent level increased after ACCF but was not altered after ACVC or ACVC-HA implantation. Compared with the control group, no significant difference was found in ROM and neutral zone (NZ) in flexion-extension or lateral bending for the ACVC and ACVC-HA groups, whereas the ROM and NZ in rotation were significantly greater. Compared with the ACCF group, the ROM and NZ significantly increased in all directions. Overall, stiffness was significantly decreased in the ACVC and ACVC-HA groups compared with the control group and the ACCF group. Similar results were found after a fatigue test of 5,000 repetitions of axial rotation. The histological results showed more new bone formation and better bone implant contact in the ACVC-HA group than the ACVC group. Goat is an excellent animal model for cervical spine biomechanical study. Compared with the intact state and the ACCF group, ACVC could provide immediate stability and preserve segmental movement after discectomy and corpectomy. Besides, HA biocoating provide a better bone ingrowth, which is essential for long-term stability. In conclusion, ACVC-HA brings new insight to treat cervical degenerative disease. # Results 

## Design of different artificial cervical joints [Fig 1](#) shows photographs of the ACVC ([Fig 1B](#)), the ACVC-HA ([Fig 1C](#)), and the titanium cage and the anterior plate ([Fig 1D](#)) implants. The ball-in-trough structure ([Fig 1A-7 and 1A-8](#)) is the most important part of this unconstrained metal-on-metal ACVC joint. In theory, the ball-in-trough articulation allows a 20° ROM in flexion-extension, a 12° ROM in lateral bending, a 360° ROM in rotation, and a 1.5-mm anterior-posterior slide horizontally. The ACVC-HA is an ACVC treated with a 30-μm hydroxyapatite biocoating via microarc oxidation technology. The titanium cage and the anterior plate were used for goat anterior cervical discectomy and fusion. The titanium cage was 8 mm wide and could be cut to a suitable length. Because the vertebral bodies of goats are much longer than those of humans, four to eight screws could be used to fix the plate and the cage. The vertebral components were hollow-structured, which allowed the surgeons to implant the autologous bone in the hollow space. Thus, the surrounding bone could grow into the vertebral components through the holes, this providing long-term stability. 

## Clinical and radiological assessments of the different prosthesis implantations in goats All surgeries were successful. Surgery time, blood loss, recovery time, and time before eating are shown in [Table 1](#). No significant differences were found in surgery time, blood loss, recovery time, and time before eating among ACVC, ACVC-HA, and ACCF groups. These findings indicate that ACVC or ACVC-HA implantation did not increase surgery time, blood loss, recovery time, and time before eating compared with the ACCF group. ::::table-wrap

10.1371/journal.pone.0178775.t001

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## Surgery characteristics of the four different groups.

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```
                      control group   ACVC group   ACVC-HA group   ACCF group
```

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Surgery time (min) 12±6 68±11 67±16 65±13
Blood loss (ml) 10±6 55±18 56±19 51±20
Recovery time (hours) 1.1±0.6 3.6±1.7 4.1±2.0 3.5±1.4
Eating time (hours) 1.7±0.5 4.5±1.8 4.5±2.2 3.9±2.3

No significant difference was found among ACVC, ACVC-HA, and ACCF
groups.:::: The goats tolerated the artificial joints very well during the experiment. All goats survived until 24 weeks after surgery. No abnormal neurological reflexes or neurological complications were found. One goat in the ACVC group and two goats in the ACCF group developed wound infections 8 to 12 days after surgery. These wound infections were cured by applying antibiotics and changing bandages daily. One goat in the ACVC-HA group and another goat in the ACCF group suffered respiratory infections and recovered by using antibiotics. No bone fracture, joint dislocation, or screw loosening were found during the experimental period. [Fig 2](#) shows the CT images of goats in the three different groups 24 weeks after surgery. In the ACVC group, the implant was properly located in the C3 vertebral body ([Fig 2B](#)). The vertebral body component of the ACVC was located in the middle of the C3 vertebral body ([Fig 2E](#)). The two lower screws were fixed to the C4 vertebral body ([Fig 2H](#)). The titanium cage was located between C2 and C4. The vertebral bodies from C2 to C4 were fixed by the plate ([Fig 2C](#)). The titanium cage was located in the trough of the C3 vertebral body ([Fig 2F](#)). The anterior plate was fixed by 3 screws to the C2 vertebral body and by 3 additional screws to the C4 vertebral body ([Fig 2I](#)). <figure> 10.1371/journal.pone.0178775.g002 <p><img src="" /></p> <figcaption>CT images of goats in control group, ACVC group, ACCF group 24 weeks after the surgery (ACVC and ACVC-HA appears similar).<br /> A, B, C, Lateral views of C2 to C4 for the three groups; D, G, transverse views of C3 and C4 vertebral bodies in control group; E, H, transverse views of C3 and C4 vertebral bodies in ACVC group; F, I, transverse views of C3 and C4 vertebral bodies in ACCF group.</figcaption> </figure> 

## Biomechanical results of the different prosthesis implantations in goats To investigate the cervical spine ROM separately, we used the three-dimensional laser optoelectronic camera system ([Table 2](#)). There were no significant differences among the control, ACVC, and ACVC-HA groups inflexion-extension and lateral bending for the C2-C6, C2-C4, C4-C5, and C5-C6 segments. The rotation ROM of the ACVC and ACVC-HA groups were greater than the ROM of the control group for the C2-C6 and C2-C4 segments. Accordingly, the ROM in rotation decreased in the ACVC and ACVC-HA groups for the C4-C5 and C5-C6 segments. For the C2-C6 and C2-C4 segments, the ROM was less for the ACCF group than for the control group in all three directions. Accordingly, the ROM of the C4-C5 segment in all three directions and the ROM of the C5-C6 segment in rotation increased in the ACCF group. ::::table-wrap
10.1371/journal.pone.0178775.t002

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## Ranges of motion profiles of the cervical segments of the four groups before the fatigue test in all directions.

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```
                  Group A          Group B         Group C          Group D
                  (Control group)   (ACVC group)     (ACVC-HA group)   (ACCF group)
```

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C2-4 (in Degrees)

Flexion-Extension 4.0±0.6 3.9±0.6 4.0±0.3 0.4±0.1[a](#),[b](#)

Lateral bending 5.9±0.7 5.6±0.8 5.8±0.5 1.3±0.3[a](#),[b](#)

Rotation 7.2±0.6 11.2±0.9[a](#) 12.3±0.7[a](#) 5.6±0.5[a](#),[b](#)

C4-5 (in Degrees)

Flexion-Extension 1.3±0.2 1.5±0.3 1.4±0.3 2.2±0.3[a](#),[b](#)

Lateral bending 1.8±0.2 2.0±0.3 2.0±0.4 2.9±0.3[a](#),[b](#)

Rotation 2.2±0.3 1.2±0.3[a](#) 1.4±0.2[a](#) 3.4±0.2[a](#),[b](#)

C5-6 (in Degrees)

Flexion-Extension 1.4±0.2 1.3±0.2 1.4±0.1 1.6±0.2

Lateral bending 2.0±0.3 1.9±0.3 2.2±0.4 2.2±0.4

Rotation 2.4±0.2 1.8±0.2[a](#) 1.9±0.3[a](#) 1.6±0.1[a](#),[b](#)

C2-6 (in Degrees)

Flexion-Extension 6.5±0.9 6.2±0.7 6.3±1.1 4.2±0.4[a](#),[b](#)

Lateral bending 9.6±1.1 9.1±1.5 9.2±0.9 6.9±0.4[a](#),[b](#)



## Rotation 11.2±0.8 14.5±1.3[a](#) 15.2±1.8[a](#) 9.8±0.8[a](#),[b](#)

no significant differences between ACVC and ACVC-HA group

^a^ P\<0.05 versus the Group A (control)

^b^ P\<0.05 versus the Group B (ACVC group) or Group C (ACVC-HA group):::: The biomechanical data of the C2-C4 segment recorded before the fatigue test ([Table 3](#), Figs [3](#) and [4](#)) confirmed that the ROM of ACVC and ACVC-HA groups in flexion-extension and lateral bending were similar to those of the control group. It also indicated that the ROM of ACVC and ACVC-HA groups in rotation increased compared with that of the control group. Also, compared with the control group, ROM in all directions was decreased in ACCF group. ::::table-wrap
10.1371/journal.pone.0178775.t003

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## Ranges of motion, neutral zones, and stiffness profiles of the C2-4 segments of the four groups before the fatigue test in all directions.

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Motion Group A Group B Group C Group D
(Control group) (ACVC group) (ACVC-HA group) (ACCF group)

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Range of Motion (in Degrees)

Flexion-Extension 6.7±1.1 6.5±0.8 6.6±0.9 0.6±0.3[a](#),[b](#)

Lateral bending 10.1±1.2 9.7±1.4 9.8±1.2 2.3±0.6[a](#),[b](#)

Rotation 10.4±0.9 18.5±1.4[a](#) 17.3±1.3[a](#) 7.4±0.6[a](#),[b](#)

Neural Zone (in Degrees)

Flexion-Extension 1.2±0.6 1.4±0.7 1.5±0.5 0.3±0.1[a](#),[b](#)

Lateral bending 1.1±0.5 1.6±0.8 1.6±0.7 0.3±0.1[a](#),[b](#)

Rotation 1.2±0.2 3.5±0.5[a](#) 3.2±0.4[a](#) 0.5±0.2[a](#),[b](#)

Stiffness (in Nm/Degree)

Flexion-Extension 0.63±0.10 0.30±0.07[a](#) 0.33±0.11[a](#) 7.20±1.33[a](#),[b](#)

Lateral bending 0.13±0.03 0.05±0.02[a](#) 0.05±0.01[a](#) 2.53±0.67[a](#),[b](#)



## Rotation 0.21±0.02 0.02±0.00[a](#) 0.02±0.00[a](#) 1.53±0.42[a](#),[b](#)

no significant differences between ACVC and ACVC-HA group

^a^ P\<0.05 versus the Group A (control)

^b^ P\<0.05 versus the Group B (ACVC group) or Group C (ACVC-HA group):::: <figure> 10.1371/journal.pone.0178775.g003 <p><img src="" /></p> <figcaption>Typical hysteresis curves of C2-C4 level of the four groups before fatigue test in the directions of flexion-extension (A), lateral bending (B), and rotation (C).</figcaption> </figure> <figure> 10.1371/journal.pone.0178775.g004 <p><img src="" /></p> <figcaption>Average unidirectional ROM (A), NZ (B), and stiffness (C) of the four different groups at 24 weeks after surgery in all directions before the fatigue test.</figcaption> </figure> There was no significant NZ difference in flexion-extension and lateral bending among control, ACVC and ACVC-HA groups. In rotation, NZ was significantly greater for ACVC and ACVC-HA groups than for the control group. For the ACCF group, NZ was significantly decreased in all directions. The stiffness was significantly decreased in all directions for the ACVC and ACVC-HA groups, while it was increased for the ACCF group. We found similar results for the ROM, NZ, and stiffness in the ACVC and ACVC-HA groups. Similar C2-C4 biomechanical data were found after the 5,000repetitions of the axial rotation fatigue test ([Table 4](#), Figs [5](#) and [6](#)). ::::table-wrap
10.1371/journal.pone.0178775.t004

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## Ranges of motion, neutral zones, and stiffness profiles of the C2 to C4 segments of the four groups after the fatigue test in all directions.

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Motion Group A Group B Group C Group D
(Control group) (ACVC group) (ACVC-HA group) (ACCF group)

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Range of Motion (in Degrees)

Flexion-Extension 6.5±0.8 6.2±0.9 6.3±1.1 0.7±0.4[a](#),[b](#)

Lateral bending 10.5±1.5 10.1±1.5 10.2±1.3 2.5±0.5[a](#),[b](#)

Rotation 10.6±1.2 18.3±1.7[a](#) 18.5±1.9[a](#) 7.0±0.3[a](#),[b](#)

Neural Zone (in Degrees)

Flexion-Extension 1.1±0.5 1.3±0.5 1.3±0.6 0.2±0.1[a](#),[b](#)

Lateral bending 1.2±0.7 1.5±0.7 1.4±0.6 0.4±0.2[a](#),[b](#)

Rotation 1.2±0.3 3.6±0.5[a](#) 3.4±0.5[a](#) 0.6±0.2[a](#),[b](#)

Stiffness (in Nm/Degree)

Flexion-Extension 0.69±0.20 0.35±0.10[a](#) 0.34±0.12[a](#) 7.47±1.45[a](#),[b](#)

Lateral bending 0.15±0.04 0.04±0.01[a](#) 0.05±0.02[a](#) 2.67±0.68[a](#),[b](#)



## Rotation 0.26±0.04 0.02±0.00[a](#) 0.02±0.00[a](#) 1.62±0.47[a](#),[b](#)

no significant differences between ACVC and ACVC-HA group

^a^ P\<0.05 versus the Group A (control)

^b^ P\<0.05 versus the Group B (ACVC group) or Group C (ACVC-HA group):::: <figure> 10.1371/journal.pone.0178775.g005 <p><img src="" /></p> <figcaption>Typical hysteresis curves of C2-C4 level of the four groups after fatigue test in the directions of flexion-extension (A), lateral bending (B), and rotation (C).</figcaption> </figure> <figure> 10.1371/journal.pone.0178775.g006 <p><img src="" /></p> <figcaption>Average unidirectional ROM (A), NZ (B), and stiffness (C) of the four different groups at 24 weeks after surgery in all directions after the fatigue test.</figcaption> </figure> The SI ROM and SI NZ are reported in [Table 5](#) and [Fig 7](#). The SI ROM of the ACVC and ACVC-HA groups were positive in flexion-extension and lateral bending but negative in rotation. The SI NZ of the ACVC and ACVC-HA group were negative in all directions. The SI ROM and SI NZ in the ACCF group were positive in all directions. ::::table-wrap
10.1371/journal.pone.0178775.t005

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## Stability index of the flexibility test before the fatigue test.

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Motion Group B (ACVC group) Group C (ACVC-HA group) Group D (ACCF group)

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Stability Index Range of Motion (in Degrees, SI ROM)\
Flexion-Extension 0.030±0.016 0.030±0.012 0.090±0.042[\*](#)
Lateral bending 0.040±0.019 0.030±0.015 0.228±0.079[\*](#)
Rotation -0.779±0.156 -0.663±0.192 0.712±0.281[\*](#)
Stability Index Neural Zone (in Degrees, SI NZ)\
Flexion-Extension -0.167±0.198 -0.250±0.122 0.250±0.076[\*](#)
Lateral bending -0.4555±0.362 -0.455±0.321 0.273±0.122[\*](#)
Rotation -1.917±0.876 -1.667±0.911 0.417±0.182[\*](#)

no significant differences between ACVC and ACVC-HA group

- P\<0.05 versus the Group B (ACVC group) or Group C (ACVC-HA group):::: <figure> 10.1371/journal.pone.0178775.g007 <p><img src="" /></p> <figcaption>Average changes in the stability index ROMs and stability index NZs from ACVC group, ACVC-HA group and ACCF group.</figcaption> </figure> 

## Histology Histology sections of the goat cervical spines showed no inflammatory response 24 weeks after surgery, as well as no significant metallic wear debris at the implant-bone interface. Newly formed bone was found around the vertebral body component of the ACVC and the self-drilling fixation screws. In the sections located at the ACVC vertebral body component level ([Fig 8A and 8C](#)), we found less new bone formation and more fibrous tissue around the implant for the ACVC group ([Fig 8A](#)) than for the ACVC-HA group ([Fig 8C](#)). In the sections located at the self-drilling fixation screw level ([Fig 8E and 8G](#)), we found significantly more new bone formation around the screws for the ACVC-HA group ([Fig 8G](#)) than for the ACVC group ([Fig 8E](#)). Moreover, the ACVC group had much more fibrous tissue around the screws than the ACVC-HA group. These findings indicate that, compared with the ACVC group, the ACVC-HA group had a significantly higher bone-implant contact (BIC) in both sections respectively located at the vertebral body component and screw level ([Fig 8I](#)), which is an appropriate index for measuring bone ingrowth into the implant. <figure> 10.1371/journal.pone.0178775.g008 <p><img src="" /></p> <figcaption>Comparison histological images of ACVC and ACVC-HA.<br /> A and B, 25X- and 100X- magnified images at the level of ACVC vertebral body component; C and D, 25X- and 100X- magnified images at the level of ACVC-HA vertebral body component; E and F, 25X- and 100X- magnified images at the level of fixation screws of ACVC; G and H, 25X- and 100X- magnified images at the level of fixation screws of ACVC-HA; I, Bone implant contact (BIC) of ACVC and ACVC-HA at vertebral body and screw level.</figcaption> </figure> The magnified images showed that bone formation around the implant surfaces was fragmentary and that a large portion of the bone tissue was separated from the implant surface by soft tissue in the ACVC group ([Fig 8B and 8F](#)). In contrast, in the ACVC-HA group, a large amount of the implant surface area was surrounded by a thin layer of cartilage on the vertebral body component of the ACVC ([Fig 8D](#)) and by extensive and continuous newly formed bone at the level of the fixation screws ([Fig 8H](#)).


Extracted trial result:
Outcome: Flexion-Extension stiffness of C2-4 (continuous)
Intervention: ACVC-HA - artificial cervical vertebra and intervertebral complex (ACVC) with an HA biocoating or ACVC
Comparator: anterior cervical corpectomy and fusion (ACCF)
Intervention group size: 8
Intervention mean: 0.34
Intervention standard deviation: 0.12
Comparator group size: 8
Comparator mean: 7.47
Comparator standard deviation: 1.45
Notes: Data is from after fatigue test. Original outcome was just "The stiffness".