3D Printing of Tricalcium Phosphate/Poly Lactic-co-glycolic Acid Scaffolds Loaded with Carfilzomib for Treating Critical-sized Rabbit Radial Bone Defects

Abstract

The rapid development of scaffold-based bone tissue engineering strongly relies on the fabrication of advanced scaffolds and the use of newly discovered functional drugs. As the creation of new drugs and their clinical approval often cost a long time and billions of U.S. dollars, producing scaffolds loaded with repositioned conventional drugs whose biosafety has been verified clinically to treat critical-sized bone defect has gained increasing attention. Carfilzomib (CFZ), an approved clinical proteasome inhibitor with a much fewer side effects, is used to replace bortezomib to treat multiple myeloma. It is also reported that CFZ could enhance the activity of alkaline phosphatase and increase the expression of osteogenic transcription factors. With the above consideration, in this study, a porous CFZ/β-tricalcium phosphate/poly lactic-co-glycolic acid scaffold (designated as "cytidine triphosphate [CTP]") was produced through cryogenic three-dimensional (3D) printing. The hierarchically porous CTP scaffolds were mechanically similar to human cancellous bone and can provide a sustained CFZ release. The implantation of CTP scaffolds into critical-sized rabbit radius bone defects improved the growth of new blood vessels and significantly promoted new bone formation. To the best of our knowledge, this is the first work that shows that CFZ-loaded scaffolds could treat nonunion of bone defect by promoting osteogenesis and angiogenesis while inhibiting osteoclastogenesis, through the activation of the Wnt/β-catenin signaling. Our results suggest that the loading of repositioned drugs with effective osteogenesis capability in advanced bone tissue engineering scaffold is a promising way to treat critical-sized defects of a long bone.

Keywords: Bone defect; Bone regeneration; Carfilzomib; Cryogenic 3D printing; β-tricalcium phosphate.

Figure 1

Schematic diagram depicting the fabrication of the 3D-printed β-tricalcium phosphate/poly lactic-co-glycolic acid carfilzomib-loaded scaffolds.

Figure 2

Fabrication of a 3D-printed β-tricalcium phosphate/poly lactic-co-glycolic acid scaffold loaded with carfilzomib (CFZ). (A-F) Scanning electron microscopy micrographs of different scaffolds at different magnification. (G) Compressive strengths of cytidine triphosphate (CTP) scaffolds and TP controls. (H) Young modulus of the CTP scaffolds and TP controls. (I) Release behavior of CFZ from CTP scaffolds in a 30-day test period. (J) Cell viability after seeding C3H10T1/2 in the scaffolds for 3 days.

Figure 3

Cytidine triphosphate (CTP) scaffolds promote osteogenic differentiation in C3H10T1/2 mesenchymal cells. (A) mRNA expression of osteogenic genes in C3H10T1/2 cells stimulated with osteogenic medium supplemented with (carfilzomib [CFZ] group) or without (control group) extracts of the CTP scaffold for 7 days. (B) Western blot results of osteogenic proteins in C3H10T1/2 cells from control and CFZ group. (C) Immunofluorescence staining of osteocalcin in C3H10T1/2 cells from control and CFZ group. (D) Alizarin red S staining of C3H10T1/2 cells stimulated with osteogenic medium supplemented with (CFZ group) or without (control group) extracts of the CTP scaffold for 14 days. Scale bar = 50 μm. *P < 0.05.

Figure 4

Cytidine triphosphate (CTP) scaffolds inhibit osteoclast formation in RAW264.7 cells. (A) mRNA expression of osteoclast formation genes in RAW264.7 cells stimulated with RANKL and supplemented with (carfilzomib [CFZ] group) or without (control group) extracts of CTP scaffold for 7 days. (B) Western blot results of Ctsk, MMP9 and NFATC1 in raw264.7 cells from control and CFZ group. (C) Immunofluorescence staining of F-actin and CTSK in raw264.7 cells stimulated with RANKL and supplemented with (CFZ group) or without (control group) extracts of CTP scaffold for 14 days. Scale bar = 50 μm. *P < 0.05.

Figure 5

Cytidine triphosphate (CTP) scaffolds activate Wnt/β-catenin signaling. (A) Western blot results of β-catenin in C3H10T1/2 cells cultured with medium supplemented with (carfilzomib [CFZ] group) or without (control group) extracts of the CTP scaffold. (B) TOP/FOP-Flash luciferase reporter assay was used to analyze the effect of CTP scaffolds on the activity of Wnt/β-catenin signaling in C3H10T1/2 cells. (C) Immunofluorescence staining of β-catenin in C3H10T1/2 cells from control and CFZ group. Scale bar = 50 μm. *P < 0.05.

Figure 6

Imaging results show cytidine triphosphate scaffolds promote bone regeneration in a rabbit long bone defect model. (A) Surgical procedure in rabbits with the scaffolds. (B) Micro-CT scan images of the defect area at 12 weeks post-surgery. (C) Bone mineral density of regenerated bone tissue. (D) BV/TV ratio of regenerated tissue. (E) Microfil angiography and micro-CT imaging results of the experimental animals. (F) Quantification of average vessel length and vessel area (G) of the experimental animals *P < 0.05.

Figure 7

Histological results show that cytidine triphosphate scaffolds promote bone regeneration in a rabbit long bone defect model. (A) H&E staining of the defect area at 12 weeks post-surgery. B: bone, F: Fibrous tissue, NB: New bone; V: Vessel). (B and E) IF analysis and quantification of CTSK in bone sections from different groups. (C and F) Immunofluorescence analysis and quantification of CD31 in bone sections from all groups. (D, G, and H) Immunofluorescence analysis and quantification of OCN and β-catenin in bone sections from different groups. Scale bar = 50 μm. *P < 0.05.

Figure 8

Schematic diagram of cytidine triphosphate (CTP) scaffolds promoting bone regeneration via activating Wnt/β-catenin pathway. (A) Schematic diagram of the preparation process of the CTP scaffold and its effect on angiogenesis and osteogenesis in the bone defect. (B) Chemical structure of carfilzomib and mechanism of carfilzomib on the canonical Wnt/β-catenin signaling.