Bone defects have become common in clinical practice, while the natural healing process is challenging because of the poor regenerative capacity and large defects. In this study, the 3D-printed PLA/HA composite scaffold containing USCs was successfully applied to a rat model of skull defect. The PLA/HA scaffold has the advantage of two kinds of scaffold. The mechanical property of the PLA/HA scaffold increased and the degradation rate improved, while the inflammatory reaction in the defect area decreased. Besides, the feasibility of combining scaffolds with USCs was proved via the in vitro experiment. At 12 weeks after treatment, effective bone regeneration indicated the good therapeutic effect of PLA/HA scaffold and USCs in tissue engineering.
Applying bone tissue engineering technology in the treatment of bone defects, such as preparing 3D-printed scaffolds carrying seed cells, has been considered as a new treatment in recent years . In a study about treatment outcomes of custom-made bioceramic implants, Staffa et al.  found that most patients recovered well after surgery with few adverse consequences, which proved the osteogenic property of HA. It has also been found that the mechanical properties of HA are not strong enough to be used in large-scale defects . Nevertheless, a single material does not have all the properties that tissue engineering requires. Therefore, different biopolymers and minerals were used to fabricate scaffolds with different properties . Zimina et al. produced PLA/HA composites and showed their potential for hard tissue engineering and restoring maxillofacial defects . Ideal scaffold materials in bone tissue engineering have higher biocompatibility, biodegradation, and mechanical strength; it is hard to achieve the purpose of bone defect repair otherwise [32, 33]. Although PLA has good biocompatibility and degradation ability, it is still deficient in mechanical strength and bone conductivity. Therefore, HA was combined with PLA in this study, and the results further proved that the mechanical performance and properties of composite scaffolds are superior to those of the materials acting independently (Table 2).
By extruding thermoplastic materials through a heated metal nozzle, the FDM fabrication produces various 3D designs . For example, FDM-technology-printed porous polycaprolactone and hydroxyapatite (PCL/HA) scaffolds support cell adhesion and proliferation and relieve the adverse effects of inflammation on murine chondrocytes . The physicochemical properties of the scaffold are crucial for the growth and differentiation of stem cells. It is believed that high porosity and interconnected pore structure of scaffolds are fundamental for bone regeneration in tissue engineering, and the optimum pore size ranges from 20 to 1500 µm. The size of the macropores of the scaffold is supposed to be >350 µm because seed cells need to migrate to the defect site and achieve bone regeneration . Different pore sizes of scaffolds can be easily acquired by changing printing parameters like nozzle diameter, moving speed, and extrusion pressure . In our study, PLA/HA composite scaffold was fabricated by FDM technology with the pore size ranging from 300 to 500 µm. The distribution and adhesion behavior of USCs in the PLA/HA group is more remarkable than that in other groups. We assume that the degradation component of PLA may reduce the pH in the environment, while alkaline HA has a buffering effect on acidic substances and provides suitable cell adhesion and proliferation conditions. Our results found that the PLA/HA composite scaffold showed better mechanical properties and cytocompatibility compared with the PLA-only scaffold (Fig. 4), in agreement with previous studies .
It seems possible to increase the mechanical properties of scaffolds by combining PLA with HA. However, we have to admit that even the maximum values of the Young’s modulus and the ultimate deformation strength for the printed scaffolds are many times less than that of bone tissue; hence, it may be that printed scaffolds are not suitable for weight bearing. In our study, printed scaffolds were not used as load-bearing materials but for a sort of osteoinductive biomaterial containing USCs to promote bone regeneration in the defect site. In previous studies, biomaterials of scaffolds have played a crucial role in promoting bone regeneration. For instance, PLA/HA implantation in mice demonstrated good tolerance and widespread ingrowth of newly formed bone tissue into the implant pores . Our in vitro results suggested that PLA/HA composite scaffolds induced a pronounced osteogenic gene expression (Fig. 5), which indicated that the PLA/HA composite scaffold enhanced the osteogenic potential of USCs.
Although different materials exhibit their ability to repair bone defects, successful bone regeneration still requires seed cells, which can differentiate into other types of cells. Seed cells have long been seeded onto scaffolds for tissue engineering, such as bone marrow MSCs, adipose-derived stem cells, muscle-derived stem cells, and embryonic stem cells [37–40]. However, the clinical application of most seed cells was limited by various factors such as limited sampling sites, insufficient donors, and ill-equipped cell homing. Similar to other adult stem cells, USCs conform to seed cells’ characteristics in terms of growth pattern and differentiation capacity, and it have been successfully used for urinary system, nerve tissue, and skeletal muscle tissue injury [11–14]. Our work showed that USCs expressed the surface marker of bone marrow MSCs, with conspicuous cell proliferation ability and multiple differentiation potential (Fig. 1). Besides, USCs have been used for the repair and reconstruction of bone defects. For example, the combination of PLGA/CS scaffolds and USCs has potential in bone regeneration since they can stimulate osteogenic differentiation of USCs and induce ingrowth of blood vessels into scaffolds . Many researchers have attempted to find methods to induce USCs into the osteogenic lineage , whereas few studies have focused on combining USCs with scaffolds suitable for tissue engineering. Furthermore, the effects of PLA/HA composite scaffolds on osteogenic differentiation of USCs have not been reported. So, we fabricated the PLA/HA composite scaffold via FDM 3D printing technology. USCs were inoculated onto scaffolds, which were subsequently transplanted into rats to analyze the osteogenic differentiation of USCs.
Our in vivo study showed that PLA/HA scaffold containing USCs repaired bone defects at the macro- and micro-level (Figs. 6–8). Whether in the results of the general observation or micro-CT images, the implantation group showed a better therapeutic effect than the control group at 12 weeks after treatment. The dimension of newly formed bone by micro-CT analysis indicated that the PLA/HA scaffolds containing USCs had the best performance in reconstructing bone defects and the fastest rate of scaffold degradation. Osteocalcin (OCN) is a biochemical marker that is produced by osteoblasts. It plays a vital role in bone-building and late osteogenesis. Compared with other groups, more OCN was expressed in PLA/HA groups, indicating that the PLA/HA scaffold further promoted osteogenic differentiation of USCs. The PLA/HA scaffold containing USCs caused more marked bone regeneration in the skull defect model than the PLA scaffold with USCs.
There were several limitations to our work. Although USCs have been successfully seeded onto PLA/HA scaffolds and applied to bone defect repair, it is still unclear whether USCs can be widely applied to other kinds of scaffolds. Also, we evaluated bone defect repair, but the weight-bearing capacity and mechanical properties of the newly formed bone in the defect area still have to be further tested. Finally, in this bone defect model, we did not analyze the mechanism of bone defect repair, which requires further investigation.