3D-printed porous zinc scaffold combined with bioactive serum exosomes promotes bone defect repair in rabbit radius

Abstract

Currently, the repair of large bone defects still faces numerous challenges, with the most crucial being the lack of large bone grafts with good osteogenic properties. In this study, a novel bone repair implant (degradable porous zinc scaffold/BF Exo composite implant) was developed by utilizing laser melting rapid prototyping 3D printing technology to fabricate a porous zinc scaffold, combining it under vacuum conditions with highly bioactive serum exosomes (BF EXO) and Poloxamer 407 thermosensitive hydrogel. The electron microscope revealed the presence of tea saucer-shaped exosomes with a double-layered membrane structure, ranging in diameter from 30-150 nm, with an average size of 86.3 nm and a concentration of 3.28E+09 particles/mL. In vitro experiments demonstrated that the zinc scaffold displayed no significant cytotoxicity, and loading exosomes enhanced the zinc scaffold's ability to promote osteogenic cell activity while inhibiting osteoclast activity. In vivo experiments on rabbits indicated that the hepatic and renal toxicity of the zinc scaffold decreased over time, and the loading of exosomes alleviated the hepatic and renal toxic effects of the zinc scaffold. Throughout various stages of repairing radial bone defects in rabbits, loading exosomes reinforced the zinc scaffold's capacity to enhance osteogenic cell activity, suppress osteoclast activity, and promote angiogenesis. This effect may be attributed to BF Exo's regulation of p38/STAT1 signaling. This study signifies that the combined treatment of degradable porous zinc scaffolds and BF Exo is an effective and biocompatible strategy for bone defect repair therapy.

Keywords: 3D printing; bone defect; bone repair; exosome; zinc scaffold.

Figure 1

Preparation of 3D-printed porous zinc scaffold. (A) 3D-printed macroscopic image of a porous zinc scaffold; (B) SEM images of the porous zinc scaffold at 30, 80, and 150 times magnification in the frontal view; (C) SEM images of the porous zinc scaffold at 30, 80, and 150 times magnification in the side view; (D) Stress-strain plot of the 3D-printed porous zinc scaffold, with compressive displacement on the x-axis and compressive load on the y-axis, where the black triangles represent the first peak compressive load; (E) In vitro cytotoxicity evaluation of the 3D-printed porous zinc scaffold.

Figure 2

Extraction and identification of exosomes from rabbit serum for biological activity. (A) The analysis of exosome particle size using nanoscale flow cytometry; (B) The analysis of exosome concentration. (C, D) Transmission electron microscopy images of exosomes in serum samples from rabbit models with femoral fractures, where C shows the exosome suspension at dilutions of 10, 30, and 50-fold from left to right, and D illustrates the protein expression of CD63, TSG101, and syntenin1 in exosomes from the serum of rabbit models with femoral fractures.

Figure 3

Preparation of zinc scaffold/BF Exo composite implant. (A) Solid images of degradable zinc stent/BF Exo composite implant; (B) Electron microscopy images of degradable zinc stent/BF Exo composite implant at 50, 200, 1000, and 2000 times magnification, with red arrows indicating small amounts of exosomes encapsulated in the gel; (C) X-ray diffraction patterns of degradable zinc stent/BF Exo composite implant; (D) CCK-8 experiment results after co-culturing BMSCs cells with degradable zinc stent/BF Exo composite implant for 3 days; (E) In vitro release results of exosomes from zinc stent/BF Exo composite implants after incubation for 1, 3, 5, and 7 days; (F) In vitro release results of zinc ions after incubating two types of stents for 1, 3, 5, and 7 days (^**P < 0.01, ^****P < 0.0001 vs. Zinc scaffold, n = 3).

Figure 4

In vitro osteogenic performance assessment of zinc scaffold/BF Exo composite implant. (A) The CCK-8 experiment was used to measure the cell viability of BMSCs after osteogenic induction for 1, 3, 5, and 7 days; (B) The CCK-8 experiment was used to assess the cell viability of RAW264.7 cells after osteoclast induction for the same time periods; (C, D) ALP staining was conducted for BMSCs after 3 and 7 days of osteogenic induction, where darker staining indicated stronger positive expression; (E, F) Alizarin Red staining was performed for BMSCs after 14 and 21 days of osteogenic induction; (G) TRAP staining was conducted for RAW264.7 cells after 7 days of osteoclast induction (^*P < 0.05, ^**P < 0.01, ^****P < 0.0001, and ns for P > 0.05, n = 3).

Figure 5

The ELISA experiment on rabbit serum. (A–E) The levels of PICP, BALP, ICTP, VEGF, and FGF-2 in the serum of rabbits were measured at 8 and 12 weeks after modeling (^*P < 0.05, ^**P < 0.01, ^****P < 0.0001, ^*P > 0.05, n = 3).

Figure 6

Fluorescence and staining experiments on rabbit radius bones. (A) Alizarin Red and Calcein Green fluorescence results of rabbit radius tissues after 8 weeks of modeling in each group; (B) Alizarin Red and Calcein Green fluorescence results of rabbit radius tissues after 12 weeks of modeling in each group; (C) Methylene blue-acidic magenta staining results of rabbit radius tissues after 8 and 12 weeks of modeling in each group (100×, n = 3).

Figure 7

Assessment of in vivo hepatorenal toxicity of zinc scaffold/BF Exo composite implant. (A) HE staining results and physical samples of rabbit liver tissue after 8 weeks of modeling by each group; (B) HE staining results and physical samples of rabbit liver tissue after 12 weeks of modeling by each group; (C) HE staining results and physical samples of rabbit kidney tissue after 8 weeks of modeling by each group; (D) HE staining results and physical samples of rabbit kidney tissue after 12 weeks of modeling by each group (200×, n=3).

Figure 8

Micro-CT imaging experiment of rabbit radius tissue. (A) Imaging results of Micro-CT of rabbit radius in each group at 8 weeks after modeling. Contains Sample, Scan, Implantation, Cambium (New bone formation) and Merge images, respectively; (B) Imaging results of Micro-CT of rabbit radius in each group at 12 weeks after modeling; (C–F) Results of quantitative analyses of BV, BV/TV, BMC, and BMD in each group of animals at 8 and 12 weeks after modeling (^*P < 0.05, ^**P < 0.01, ^****P < 0.0001, ^nsP > 0.05 vs. Zinc scaffold, n = 3).

Figure 9

Immunohistochemical analysis of in vivo implantation of zinc scaffold/BF Exo composite implant. (A) Immunohistochemical staining results of rabbit radius in each group after 12 weeks of modeling; (B) Quantitative analysis results of p38 in rabbit radius in each group after 12 weeks of modeling; (C) Quantitative analysis results of STAT1 in rabbit radius in each group after 12 weeks of modeling (^*P < 0.05, ^****P < 0.0001, ^nsP > 0.05 vs. Zinc scaffold, n = 3).