Supplementary Materials and methods

Cervical cage design and manufacturing optimization

Data collection and processing

The original CT images of the cervical spine were collected in DICOM format. The cervical vertebrae parameters were measured by PACS system through CT imaging. After importing the parameters into Mimics 18.0 (Materialise, Leuven, Belgium), the bone tissue was separated by the “Thresholding tool” and the region extended by the “Region growing” tool. The “Edit mask” tool was used to separate different segments of the cervical spine (labeled in a different color). The targeted cervical segments (C3/C4) were reconstructed by the “Calculate 3D” tool. The “Morphology operation” tool was used to fill small voids and smoothe the surface of the bony structure. (Supplementary Figure 2 a-b)

Cage design and optimization

The structure of the targeted cervical intervertebral space can be obtained by using the established digital model, which can be substantiated by using Rhino 5.0 (Robert McNeel & Assoc.). First, a 15 mm × 15 mm × 10 mm intervertebral disc cage model and a cervical vertebra model were imported into Rhino 5.0 and adjusted to the appropriate position. Second, the redundant portion of the cage was subtracted by “Boolean calculation,” so that the upper and lower surfaces of the cage completely match the cervical space. The cage was then divided, along with the connected vertebra, into four equal sections to verify their fit. Finally, the modified cage was moved out of the vertebra to form the bone graft hole by Boolean subtraction calculation. (Supplementary Figure 2 c-g)

Cage manufacturing

For the manufacturing process, an FDM printer (Flashforge Dreamer, NY, US) was used to fabricate the 3D structure with a layer-by-layer deposition of polymer. Following the design and optimization, the cage model was transferred to 3D printing in an STL file and the related parameters were adjusted. The PLA and 20% pβ-TCP composites were used to print cages. The printing precision was 0.1 mm. (Supplementary Figure 2 h)

Cell culture

MC3T3-E1 cells, a mouse osteoblastic cell line, were obtained from the cell bank of the Chinese Academy of Sciences. MC3T3-E1 cells were cultured in α-MEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in 5% CO2 at 37 °C. The medium was changed three times a week.

Cell Counting Kit-8 (CCK-8) test

The pure PLA materials and PLA/pβ-TCP composite materials were printed into layers using a 3D printer, and cut into 1 cm × 1 cm strips using scissors. After sterilization, the materials were placed in a 24-well plate. 1 × 105 cells dissolved in 30-µl medium were seeded on the materials, and showed adherence after incubation overnight. A total of 1 ml of α-MEM medium was added to the wells and cultured for 1, 4, and 7 days. The medium was replaced every 2 days. At days 1, 4, and 7, the culture medium was discarded, and 500 µl fresh α-MEM containing 10% (v/v) CCK-8 was added and incubated for 2 h. Then, 100 µl of this reaction solution was added into a new 96-well plate. The OD value was measured using a microplate reader at a wavelength of 450 nm. Each sample was run in two sets of triplicate (n = 6).

Supplementary Table 1. The indexes of C3-C4 in nine cervical spines (cm)

N. / Upper VSD (cm) / Upper VHD (cm) / Lower
VSD (cm) / Lower
VHD (cm) / Internal
VHD (cm) / Central
VH (cm) / Peripheral VH (cm)
1 / 2.06 / 1.84 / 2.08 / 1.92 / 2.45 / 0.72 / 0.88
2 / 2.15 / 2.04 / 2.38 / 2.12 / 2.93 / 0.74 / 0.92
3 / 2.16 / 1.54 / 2.44 / 1.93 / 2.71 / 0.78 / 0.80
4 / 2.11 / 1.81 / 2.41 / 2.01 / 2.62 / 0.71 / 0.95
5 / 2.27 / 1.77 / 2.47 / 2.04 / 2.89 / 0.75 / 0.84
6 / 2.17 / 1.78 / 2.31 / 2.06 / 2.52 / 0.72 / 0.9
7 / 2.14 / 1.75 / 2.50 / 2.09 / 2.84 / 0.74 / 0.9
8 / 2.15 / 1.89 / 2.62 / 2.07 / 2.88 / 0.65 / 0.78
9 / 2.07 / 1.70 / 2.31 / 2.04 / 2.53 / 0.63 / 0.79
Mean
(cm) / 2.14 ± 0.06 / 2.39 ± 0.15 / 1.79 ± 0.14 / 2.03 ± 0.07 / 2.71 ± 0.18 / 0.72 ± 0.05 / 0.86 ± 0.06

VSD: vertebral sagittal diameter; VHD: vertebral horizontal diameter; VID: vertebral internal diameter; VH: vertebral height

Supplementary Figure 1 Histogram of cell viability via CCK-8 assay. The data are representative of three independent experiments. Error bars represent mean ± SD *P 0.05, by Student’s t-test.

Supplementary Figure 2 Design and fabrication of 3D printed cage. (a) CT data were input into Mimics 18.0 and cervical segments were marked in different colors; (b) Targeted C3/C4 segments were detached via Rhino 5.0; (c) The cage guide plate was put into and adjusted to the suitable position in the gap of C3-C4 segments; (d) The cage guide plate was trimmed by Boolean Difference Set Operation; (e) The guide plate was divided into four parts to verify the matching degree in the sagittal plane; (f) The modified cage model; (g) The formation of bone graft hole via Boolean Difference Set Operation; (h) The 3D printed PLA/β-TCP cage.