Nano-sized titanium alloy particles inhibit the proliferation and promote the apoptosis of bone marrow mesenchymal stem cells in vitro

Abstract

Aseptic loosening of artificial joints is the leading cause of failure for patients who receive total joint arthroplasty. Prior reports indicate that bone marrow mesenchymal stem cells (BSMC) are critical in the stabilization of implanted artificial joints, and that deregulated interaction between BMSCs and artificial joint derived particles is a risk factor for aseptic loosening with an unknown mechanism. In the present study, the pathomechanisms whereby titanium and its alloy derived particles facilitate aseptic loosing were investigated in vitro. It was demonstrated that nano‑sized titanium alloy particles significantly inhibited the proliferation of BMSCs in a time and concentration dependent manner. Furthermore, it was demonstrated that the particles promoted the apoptosis of BMSCs in the same manner. Bax and Caspase‑3 expression of BMSCs were elevated when cultured with the particles. As BMSCs exhibit a critical role in the stabilization of artificial joints, the results of the present study may provide a novel direction for the management of aseptic loosening in clinics.

Figure 1.

Preparation of Ti-alloy particles and BMSC. (A) Microscopic inspection showed that Ti-alloy particles were ball in shape with a diameter of less than 100 nm, the red arrow pointed was Ti-alloy particle under the microscope; (B-E) Microscopic inspection of (B) primary BMSC, (C) the first, (D) the second and (E) the third generation of BMSC; (F-H) flow cytometry assay used to confirm the phenotype of the isolated cells based on their specific cell surface markers, such as (F) CD90, (G) CD105 and (H) CD34. BSMC, bone marrow mesenchymal stem cells.

Figure 2.

Microscopic inspection toward bone marrow mesenchymal stem cell morphology when they were cultured with Ti-alloy particles at different time (6 and 72 h).

Figure 3.

Ti-alloy particles inhibit the proliferation of BMSCs in a manner dependent on time and concentration. (A) CCK8 assay reveal that Ti-alloy particles inhibit BMSC proliferation in a time dependent manner; (B) CCK8 assay reveal that Ti-alloy particles inhibit BMSC proliferation in a concentration dependent manner. *P<0.05 and ***P<0.001. BSMC, bone marrow mesenchymal stem cell. CCK8, Cell Counting Kit-8.

Figure 4.

Ti-alloy particles enhance the apoptosis of BMSCs in a manner dependent on time and concentration. (A) Flow cytometry inspection reveals that Ti-alloy particles promote BMSC apoptosis in a time and concentration-dependent manner (the upper left quadrant was the total collected cells for TCM assay, the lower left and right quadrants indicated the apoptosis rate of BMSC at the early/late stage, their product was the total apoptosis rate of BMSCs). (B) Statistical analysis suggested that with the particles concentration grows, its role in promoting BMSCs apoptosis was becoming more and more prominent. In addition, with the co-culture time grows, the role of the particles in promoting BMSCs apoptosis was also becoming increasingly prominent. (C) Immunofluorescence staining assay revealed that Ti-alloy particles promote BMSC apoptosis in a time and concentration dependent manner. *P<0.05; **P<0.01, and ***P<0.001. BSMC, bone marrow mesenchymal stem cell.

Figure 5.

Ti-alloy particles up regulate Caspase-3 and Bax expression of BMSCs in a manner dependent on time and concentration. (A and B) RT-PCR showed that Caspase-3 expression of BMSC was upregulated in a time and dependent concentration manner when cultured with Ti-alloy particles. (C and D) RT-PCR showed that Bax expression of BMSC was upregulated in a time and dependent concentration manner when cultured with Ti-alloy particles. (E and F) Western blot assay revealed that Ti-alloy particles promote Caspase-3 expression in time dependent manner in protein levels. (G and H) Western blot assay revealed that Ti-alloy particles promote Bax expression in a concentration dependent manner in protein levels. Relative densities are presented as mean 6 SE (n=3) of the fold-change relative to the internal control. GAPDH was used as an internal control for protein loading. *P<0.05; **P<0.01, and ***P<0.001. BSMC, bone marrow mesenchymal stem cell.

Figure 6.

The schematic picture that depicts the overview of the article. As depicted, the artificial implants derived particles could permeate into the bone marrow. Then, the filtrated particles inhibit the proliferation and promote the apoptosis of BMSC, a critical component that involve in the stabilization of the implants. BSMC, bone marrow mesenchymal stem cell.