3D Printing Surgical Implants at the clinic: A Experimental Study on Anterior Cruciate Ligament Reconstruction

Abstract

Desktop three-dimensional (3D) printers (D3DPs) have become a popular tool for fabricating personalized consumer products, favored for low cost, easy operation, and other advantageous qualities. This study focused on the potential for using D3DPs to successfully, rapidly, and economically print customized implants at medical clinics. An experiment was conducted on a D3DP-printed anterior cruciate ligament surgical implant using a rabbit model. A well-defined, orthogonal, porous PLA screw-like scaffold was printed, then coated with hydroxyapatite (HA) to improve its osteoconductivity. As an internal fixation as well as an ideal cell delivery system, the osteogenic scaffold loaded with mesenchymal stem cells (MSCs) were evaluated through both in vitro and in vivo tests to observe bone-ligament healing via cell therapy. The MSCs suspended in Pluronic F-127 hydrogel on PLA/HA screw-like scaffold showed the highest cell proliferation and osteogenesis in vitro. In vivo assessment of rabbit anterior cruciate ligament models for 4 and 12 weeks showed that the PLA/HA screw-like scaffold loaded with MSCs suspended in Pluronic F-127 hydrogel exhibited significant bone ingrowth and bone-graft interface formation within the bone tunnel. Overall, the results of this study demonstrate that fabricating surgical implants at the clinic (fab@clinic) with D3DPs can be feasible, effective, and economical.

Figure 1. Schematic diagrams of the implant and tendon graft within the bone tunnel in ACL reconstruction.

(A) The 3D perspective of the bone tunnel in ACL reconstruction. (B) The transverse section view of the bone tunnel. (G: Graft; S: Screw-like scaffold; BT: Bone tunnel; M: Macropore).

Figure 2. Study design in vivo.

The screw-like scaffold was designed and fabricated by the D3DP. All rabbits were randomly divided into PLA group (PLA scaffold implantation, n = 12), PLA/HA group (PLA/HA scaffold implantation, n = 12) and MSCs group (PLA/ HA scaffold loaded MSCs, n = 12). Complete sharp transections were established in the anterior cruciate ligament of adult New Zealand male rabbits. The long digital extensor tendon (2 mm in diameter and 3 cm in length) was harvested as tendon graft. The scaffold was pressed into the femoral tunnel to fix the tendon graft. The rabbits were sacrificed at weeks 4, 12 for subsequent analysis.

Figure 3. The sketch map of micro-CT evaluations.

(A) Gross observation of the vertical plane of the rabbit knee joint. (B) The vertical plane of the axis of the femoral bone tunnel in a sagittal view of the micro-CT image, the region of interest (ROI) was shown with new bone within the bone tunnel (white rectangle) and the tendon graft can be observed (white triangle). (C) The cross section of the axis of the femoral bone tunnel of the micro-CT image, ROI was shown with new bone within the bone tunnel (white circle). (D) The external aperture of the femoral bone tunnel in a 3D reconstruction micro-CT image (black star), new bone growth can be seen within the bone tunnel.

Figure 4. Physical properties of the PLA screw-like scaffold.

(A) 3D view of the theoretical designed PLA screw-like scaffold structure. (B) The prepared PLA screw-like scaffold. (C) The SEM image of the PLA scaffold surface with well-defined orthogonal structure.

Figure 5. Characterization of synthesized hydroxyapatite (HA).

(A) The SEM image of HA crystals. (B) The XRD spectrum of HA crystals.

Figure 6. SEM images of the PLA/HA scaffold.

(A) The modification of HA on the 3D structure of the PLA scaffold. (B) The closer observation of the HA modification on the PLA surface.

Figure 7

SEM images of the MSCs seeded on (A) PLA scaffold, (B) PLA/HA scaffold and (C) suspended in Pluronic F-127 solution on PLA/HA scaffold for 48 hours. The photographs (D–F) are from closer observation of MSCs, respectively.

Figure 8. CCK-8 assay for MSCs proliferation on PLA scaffold, PLA/HA scaffold, and suspended in Pluronic F-127 solution on PLA/HA scaffold at 1day, 4 days and 7 days.

^##vs. PLA scaffold, p < 0.01; ^△vs. PLA/HA scaffold, p < 0.05; ^△△vs. PLA/HA scaffold, p < 0.01; n = 5.

Figure 9

RT-PCR analysis for the osteoblast phenotypic marker genes: the level of (A) Col I, (B) OCN, (C) Sp7 and (D) Runx2 mRNA in MSCs seeded on PLA scaffold, PLA/HA scaffold, and suspended in Pluronic F-127 solution on PLA/HA scaffold at 7 days. ^##vs. PLA scaffold, p < 0.01; ^△vs. PLA/HA scaffold, p < 0.05; ^△△vs. PLA/HA scaffold, p < 0.01.

Figure 10

Images of MRI examination in MSCs groups: (A) transverse, (B) coronal and (C) sagittal section at 4 weeks; (D) transverse, (E) coronal and (F) sagittal section at 12 weeks. (G: Graft; S: Screw-like scaffold; F: Femur; T: Tibia; P: Patella).

Figure 11. 3D reconstruction micro-CT images of new bone formation within the femoral bone tunnel.

At 4 weeks, the volume of new bone growth in the MSCs group (C) was similar to that in the PLA/HA group (B), but more than that in the PLA group (A); at 12 weeks, the new bone was well distributed and interconnected in the MSCs group (F) and the volume of its new bone formation was more than that in the PLA/HA group (E) which is similar to that in the PLA group (D).

Figure 12. Histology images in the rabbit ACL reconstruction model.

At 4 weeks, there was full of fibrous tissue with less new bone formation was found at the interface tissue in the PLA group (A). The interface tissue contains more chondrocytes and cartilage matrix in the MSCs group (C) than in the PLA/HA group (B); At 12 weeks, the spaces between the tendon graft and the bone tunnel were narrower in all three groups (D–F). Compared with the PLA group (D) and PLA/HA group (E), the tendon graft side was in intimate contact with the new bone and increased collagen fiber continuity between the new bone and the tendon in the MSCs group (F). (B: Bone; IF: Interface; T: Tendon graft; S: Screw-like scaffold; Von-Gieson stain, original magnification ×100).

Figure 13. The concept map of the fab@clinic D3DP.

(A) Patients who suffer trauma went to the hospital for treatment. (B) Patients get consultation in hospital and do radiology examination. (C) With the help of open source online and radiologic data, surgeons design and reconstruct customized surgical implants in the office. (D) Surgeons, company mangers and sellers get together to develop the fab@clinic D3DP. (E) The customized implants are fabricated with the fab@clinic D3DP in the surgeons’ office. (F) The customized implants are implanted during the operation. (G) Since fab@clinic D3DPs are widely applied in surgery, the sales volume will increase rapidly to achieve a big market and create considerable economic value. Figure 13 was drawn by Miao Sun.