Early recovery of weight-bearing function and reconstruction of complex geometries by regenerated autologous bone are the key goals of repairing complex anatomical weight-bearing bone defects. The ideal repair is one in which shape-matched materials provide sufficient mechanical support and induce new bone to grow quickly and fully during the degradation process. However, currently used degradable materials do not guarantee mechanical support as they degrade. In this study, to achieve bone defect repair with as much autologous bone growth as possible under the condition of early load bearing, we prepared a macroporous Ti6Al4V frame with a defect-matching shape by 3D printing to provide short-term mechanical support and filled the frame with osteogenic biomaterial prepared by SECCS to achieve long-term bone repair by regenerated bone. This strategy is a new approach for repair of complex anatomical weight-bearing bone defects with both short-term and long-term benefits.
Additive manufacturing enables design freedom and manufacturing flexibility, so it has unparalleled advantages in the preparation of customised implants . EBM, by virtue of its higher-energy density, has become the main method of additive manufacturing for metal implant fabrication in recent years . Metal implants prepared by EBM have been used in spinal intervertebral fusion cages, upper cervical vertebral and skull reconstruction [14–16]. To enhance the long-term stability of implants, most of those prepared by EBM have had a microporous structure to provide the potential for long-term osseous ingrowth. However, the pore size of the microporous structures reported so far have mostly ranged between 100–500 µm, and the volume of bone that can grow is very limited . Therefore, the bony holding force produced by this limited new growth mass is restricted. In this study, we tried to replace the widely used microporous structure with a large-aperture frame structure to leave more space for bone growth. To select the appropriate printing parameters to ensure the mechanical strength of the frame, we evaluated the mechanical properties of the cylindrical frame with three sets of parameters according to the mechanical test standard . Inspired by a honeycomb, in which each comb is connected to other combs on all six sides and achieves good space utilisation with less material, we used a regular hexagonal basic printing unit. The width of the side and diameter of the inscribed circle of the hexagonal units are the two key factors that affect the overall mechanical properties of the frame. Theoretically, a larger inscribed circle diameter provides more space for bone filling but reduces the mechanical strength, whereas the opposite relationship is observed for the width of the side. In this study, the frame with the combination of w = 1.9 mm and d = 4.4 mm for the hexagonal units had better mechanical performance, indicating that the mechanical increment caused by the increase in the side width from 1.7 mm to 1.9 mm exceeded the mechanical decrement caused by the increase in the inscribed circle diameter from 4.0 mm to 4.4 mm. Additionally, the mechanical increase by increasing the side width from 1.9 mm to 2.0 mm did not exceed the mechanical decrease by increasing the inscribed circle diameter from 4.4 mm to 5.0 mm. Consequently, we used the optimal width and diameter parameters to prepare the Ti6Al4V frame, which matched the lateral half of the goat distal femur. The corresponding internal fixation system was designed and fabricated at the same time. Since the defect model involved the articular surface, to minimise potential joint damage, a PCL film was used to cover the frame’s distal end to improve smoothness and reduce rigidity. In addition, a Bio-gide collagen membrane was sutured into the surface of this PCL film to further protect the joints.
The osteogenic ability of bone fillers is the key to bone defect repair. Since autologous bone grafting, which currently is the gold standard for bone grafting treatment, has some disadvantages, such as limited supply and bone-collecting–related complications, finding bone substitutes with similar osteogenic ability has always been an important goal in orthopaedic research . Bone marrow MSCs are indispensable for bone repair [20, 21]. A large number of studies have confirmed that use of MSCs with bone substitutes can effectively promote bone repair effect [22–24]. The SECCS we developed previously can rapidly incorporate processes of screening, enrichment of autologous bone marrow MSCs and generation of composites with porous bone substitutes intraoperatively. These composites have shown superior osteogenic effects in animal models and clinical patients [10, 25]. In this study, the number of MSCs in bone marrow was significantly decreased after treatment with SECCS, indicating that MSCs successfully adhered to the porous β-TCP. From the testing particles, the adhered MSCs were widely distributed on the inner wall of the β-TCP and fully spread. The number of bone marrow MSCs replanted is an important factor in determining the effect of bone repair [26, 27]. In this study, each goat was replanted with about 31,321.7 ± 22,554.7 MSCs on average in the MSCs/β-TCP–filling group. In the reported studies on bone tissue engineering, most of the implanted MSCs were obtained through in vitro culture, and the number of transplanted MSCs often reached millions, which was much higher than that in this study [22, 23]. In this regard, we believe that SECCS has the following advantages over in vitro culture technology that outweigh its relatively lower number of replants. First, although the number of MSCs expanded in vitro was huge, they originated from a small number of primary cells. In terms of replanted primary MSCs, SECCS showed an overwhelming advantage. Since MSCs will inevitably undergo replicative senescence during in vitro culture, the proliferation and differentiation ability of MSCs will be reduced to a certain extent after expansion, whereas the MSCs replanted by SECCS retain their original ability [28, 29]. Second, the bone marrow MSCs expanded in vitro experienced sudden changes in the living environment after implanting into the body, which potentially could partially inhibit cell viability. However, the cells replanted by SECCS were not cultured in vitro, circumventing this inhibitory factor.
The lateral half of the goat distal femurs had both partial weight-bearing function and complex geometry and was easy to expose and observe. Therefore, it was used as a bone defect model in this study. Intraoperative photos and postoperative X-rays showed that the frame prepared by 3D printing completely matched the defect shape, and the shape was still well maintained 9 months after surgery. Postoperative X-ray radiograms showed that the scattered particles around the frame in the MSCs/β-TCP–filling group had degraded and disappeared by 3 months after surgery, whereas those in the β-TCP–filling group were still visible at 3 months after surgery, suggesting that the former had a relatively faster degradation rate than that of the latter, which is consistent with the results found in our previous study . In addition, the bony bridging sign at the proximal fracture site was seen earlier in the MSCs/β-TCP–filling group than in the β-TCP–filling group, which may have been related to the recruitment of autologous MSCs by implanted MSCs to promote local bone ingrowth [31, 32]. From the CT results, even though we rounded the distal end of the frame, there was still joint damage, such as patella displacement and joint wear. This finding may be because the end of the frame damaged the outer protective complex during the wear process. Therefore, for defects with joint retention, it may be more appropriate to adopt a frame with a non-porous and polished surface. In terms of defect repair, the CT results showed that tissue ingrowth was present even in the frames without material filling, suggesting that a microporous structure may not be a necessary condition for tissue ingrowth. Because of the interference by metal artefacts, we could not evaluate the tissue composition on the ground of the grey value of the tissue within the frame.
To observe the osteogenesis effect inside the frame, we performed segmented slices of the repaired frame. A better bone construction effect within the frame could be seen at each section in the MSCs/β-TCP–filling group, with new trabecular bone tightly integrated with the frame wall and the Harval system under reconstruction, which suggests that the new bone has good plasticity. Generally, the new bone area at the distal section accounts for a relatively large proportion. This characteristic may be due to the larger contact area between the distal part and contralateral normal bone, which results in a more complete local osteogenesis environment. Studies have shown that the degradation cycle of porous β-TCP is between 6 to 18 months. In this study, the porous β-TCP with a diameter of 1–3 mm implanted in the defect region had been almost degraded by 9 months after surgery, which is consistent with previous reports. Interestingly, in the pure β-TCP–filling group, a small new bone island appeared at the centre of the filling area, which suggests that the new bone formation mode mediated by porous materials did not completely depend on the gradual replacement from the surrounding normal bone to the material pores. The ossification process may also occur independently inside the porous material, possibly through the penetrated bone marrow. Although the CT results indicated that there was tissue ingrowth in the three groups, the amount of new bone in the blank group and β-TCP–filling group was very limited, mainly manifested as fibrous scar tissue. Accordingly, the osteogenic capacity of the filler significantly affected the long-term osteogenic effect, and the MSCs/β-TCP prepared by SECCS exhibited better osteogenic performance in this defect model.
In this study, in response to the clinical problem of complex anatomical bone defects in weight-bearing areas, we proposed and assessed an approach to achieving bone repair by providing a larger volume of final autologous bone; this approach involved preparing a large-aperture weight-bearing frame by 3D printing and placement of biomaterials modified by SECCS in the frame to achieve long-term bone filling. This approach may provide a new treatment option for clinical repair of such bone defects.