Dual-functional porous and cisplatin-loaded polymethylmethacrylate cement for reconstruction of load-bearing bone defect kills bone tumor cells

Abstract

Malignant bone tumors are usually treated by resection of tumor tissue followed by filling of the bone defect with bone graft substitutes. Polymethylmethacrylate (PMMA) cement is the most commonly used bone substitute in clinical orthopedics in view of its reliability. However, the dense nature of PMMA renders this biomaterial unsuitable for local delivery of chemotherapeutic drugs to limit the recurrence of bone tumors. Here, we introduce porosity into PMMA cement by adding carboxymethylcellulose (CMC) to facilitate such local delivery of chemotherapeutic drugs, while retaining sufficient mechanical properties for bone reconstruction in load-bearing sites. Our results show that the mechanical strength of PMMA-based cements gradually decreases with increasing CMC content. Upon incorporation of ≥3% CMC, the PMMA-based cements released up to 18% of the loaded cisplatin, in contrast to cements containing lower amounts of CMC which only released less than 2% of the cisplatin over 28 days. This release of cisplatin efficiently killed osteosarcoma cells in vitro and the fraction of dead cells increased to 91.3% at day 7, which confirms the retained chemotherapeutic activity of released cisplatin from these PMMA-based cements. Additionally, tibias filled with PMMA-based cements containing up to 3% of CMC exhibit comparable compressive strengths as compared to intact tibias. In conclusion, we demonstrate that PMMA cements can be rendered therapeutically active by introducing porosity using CMC to allow for release of cisplatin without compromising mechanical properties beyond critical levels. As such, these data suggest that our dual-functional PMMA-based cements represent a viable treatment option for filling bone defects after bone tumor resection in load-bearing sites.

Keywords: Bone tumor treatment; Chemotherapeutic drug; Ex vivo biomechanical assessment; Local drug delivery; Porous polymethylmethacrylate cement.

Graphical abstract

Fig. 1

Mechanical properties of PMMA-based cements containing different amounts of CMC. (A) Compressive (offset) yield strength, (B) elastic modulus of PMMA-based cements containing up to 4% CMC content and (C) fracture toughness of PMMA-based cements containing up to 4% CMC content. An asterisk indicates statistically significant differences (*P < 0.05). Error bars represent standard deviations. At least four samples of each group (n ≥ 4) are used for all mechanical studies.

Fig. 2

Setting time and maximum temperature during setting of PMMA-based cements containing 0–4% CMC. (A) Final setting time of PMMA-based cements and (B) maximum temperature at the center of the PMMA-based cement specimens. Error bars represent standard deviations. Four samples of each group (n = 4) are used for the quantitative analysis.

Fig. 3

Ex vivo biomechanical evaluation of compressive strength of tibias filled with PMMA-based cements containing up to 4% CMC. (A) Representative photographs of compressive testing of tibias filled with PMMA-based cements including intact and defected tibias as controls, (B) ultimate compressive strength and (C) elastic modulus of different experimental groups. An asterisk indicates statistically significant differences (*P < 0.05). Error bars represent standard deviations. Four samples of each group (n = 4) are used for all quantitative studies.

Fig. 4

Representative micro-CT images and reconstructions of PMMA-based cements containing different amounts of CMC. (A–D) 2D micro-CT X-ray images of longitudinal sections, (E–H) 2D micro-CT X-ray images of transversal cross-sections and (I–L) 3D reconstructions of PMMA-based cements with different CMC content (1–4%). The colorful shapes above display the plenty of pores in the porous PMMA specimens. All specimens have a cross-sectional diameter of 6 mm. Scale bars correspond to 1 mm.

Fig. 5

Quantitative analysis of porosity of PMMA-based cements with different amounts of CMC content. (A) Total porosity of PMMA-based cements, (B) percentage of open porosity, (C) percentage of closed porosity, (D) the ratio of object surface and volume and (E) percentage of accessible porosity of PMMA-based cements. (A–E) were analyzed and calculated by using micro-CT and Amira-Avizo 3D Software. An asterisk indicates statistically significant differences (*P < 0.05). Error bars represent standard deviations. Four samples of each group (n = 4) are used for all quantitative porosity studies.

Fig. 6

Surface morphologies of PMMA + cisplatin and PMMA+4%CMC + cisplatin. Representative scanning electron micrographs of the surface (A–B), cross sectional area (C) and elemental mapping images (D) of PMMA + cisplatin; Representative scanning electron micrographs of the surface (E–F), the cross sectional area (G) and elemental mapping images for barium (dark red) and platinum (green) (H) of PMMA+4%CMC + cisplatin. Scale bars correspond to 300 μm in all the micrographs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

In vitro Pt release kinetics of cisplatin-loaded PMMA-based cements with different CMC content. Cumulative Pt release percentages of cisplatin-loaded PMMA-based cements containing up to 4% CMC in (A) the first 24 h, and (B) a period of 28 days. All the specimens were immersed in HEPES buffer (pH = 6.5). Error bars represent standard deviations. Four samples of each group (n = 4) are used for all quantitative studies.

Fig. 8

Effects of Pt release from PMMA-based cements with different CMC content (1–4%) on cultured MG-63 osteosarcoma cells. (A) Quantitative analysis of the cytotoxic effects of the released Pt species on cell proliferation determined by the CCK-8 assay, (B) representative fluorescence images of the cytotoxic effects of the releasates from cisplatin-loaded PMMA-based cement containing 4% CMC and free cisplatin (positive control: 0.8 μM) on cell viability assessed by live/dead staining and (C) quantitative analysis of the percentage of the dead cells based on live/dead staining. All experiments are performed at scheduled time points (1, 4 and 7 days). An asterisk indicates statistically significant differences (*P < 0.05). Error bars represent standard deviations. Four samples of each group (n = 4) are used for all quantitative studies. Scale bars correspond to 100 μm.