Biological evaluation of three-dimensional printed co-poly lactic acid/glycolic acid/tri-calcium phosphate scaffold for bone reconstruction

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Objective: To biologically evaluate the three-dimensional(3D) printed co-poly lactic acid/glycolic acid/tri-calcium phosphate(PLGA/TCP) scaffold which could be used for repairing oral and maxillofacial bone defects, and to provide experimental evidence for its further research and clinical application. Methods: PLGA/TCP scaffolds were fabricated using low temperature rapid prototyping technique. Micro-CT and scanning electron microscope(SEM) were used to characterize the surface morphology. MC3T3-E1 cells were seeded onto the scaffold and stained with the rhodamine phalloidin and calcein acetomethoxy. After that, confocal laser scanning microscope was exploited to observe the features and viability of the cells. Moreover, the cells were co-cultured with the extract of PLGA/TCP and complete medium, respectively. The proliferation capability of the cells was assessed by the cell counting kit-8 (CCK-8) on the 1st, 2nd, and 3rd day. The PLGA/TCP scaffolds incorporated with recombinant human bone morphogenetic protein-2(rhBMP-2) of 0, 30, 60 μg(i.e. blank control group, low-dose group and high-dose group) were implanted into the latissimus dorsi muscle of the rats, and 6 weeks later, the samples were harvested to estimate the volume and pattern of new bone. Results: The 3D printed PLGA/TCP scaffold possessed a regular and well-defined porous stereo-structure with porosity of (73±3)%. Micro-CT and SEM showed that pore size were (379±32) and (453±29) μm respectively, and distance between layers were (452± 24) and (415±25) μm, and cylinder diameter were (342±24) and (350±28) μm. It also exhibited excellent cell adhesion and growth ability on the exterior and inner surface through rhodamine phalloidin and calcein acetomethoxy staining. The CCK-8 test demonstrated that the absorbance value of extract group on the 1st and 2nd day(0.51±0.08 and 0.63±0.09) were significantly higher than those in the blank control group(0.39± 0.05 and 0.53±0.05)(P<0.05), while there was no significant difference between the extract group(0.67±0.06) and the blank control group(0.68±0.04)(P>0.05) on the 3rd day. For in vivo test, there was obvious ectopic new bone formation on the PLGA/TCP scaffold incorporated with rhBMP-2, and this was demonstrated using the histological examination and micro-CT. The bone formation in the low-dose group was similar to the shape of the pre-implanted 3D printed scaffold, while much diversity was revealed in the high-dose group duo to over osteogenesis which was validated by the examinations of gross observation, histology and micro-CT. Conclusions: Customized PLGA/TCP scaffolds can be manufactured by 3D printing technique. The scaffold showed an excellent biocompatibility and ectopic osteogenesis when incorporated with rhBMP-2. However, further research is needed to validate it's effect on repairment of the oral and maxillofacial bone defects.