Novel Tuning of PMMA Orthopedic Bone Cement Using TBB Initiator: Effect of Bone Cement Extracts on Bioactivity of Osteoblasts and Osteoclasts

Abstract

Bone cement containing benzoyl peroxide (BPO) as a polymerization initiator are commonly used to fix orthopedic metal implants. However, toxic complications caused by bone cement are a clinically significant problem. Poly (methyl methacrylate) tri-n-butylborane (PMMA-TBB), a newly developed material containing TBB as a polymerization initiator, was found to be more biocompatible than conventional PMMA-BPO bone cements due to reduced free radical generation during polymerization. However, free radicals might not be the only determinant of cytotoxicity. Here, we evaluated the response and functional phenotypes of cells exposed to extracts derived from different bone cements. Bone cement extracts were prepared from two commercial PMMA-BPO cements and an experimental PMMA-TBB. Rat bone marrow-derived osteoblasts and osteoclasts were cultured in a medium supplemented with bone cement extracts. More osteoblasts survived and attached to the culture dish with PMMA-TBB extract than in the culture with PMMA-BPO extracts. Osteoblast proliferation and differentiation were higher in the culture with PMMA-TBB extract. The number of TRAP-positive multinucleated cells was significantly lower in the culture with PMMA-TBB extract. There was no difference in osteoclast-related gene expression in response to different bone cement extracts. In conclusion, PMMA-TBB extract was less toxic to osteoblasts than PMMA-BPO extracts. Although extracts from the different cement types did not affect osteoclast function, PMMA-TBB extract seemed to reduce osteoclastogenesis, a possible further advantage of PMMA-TBB cement. These implied that the reduced radical generation during polymerization is not the only determinant for the improved biocompatibility of PMMA-TBB and that the post-polymerization chemical elution may also be important.

Keywords: arthroplasty; benzoyl peroxide; cytotoxicity; implant; poly(methyl methacrylate); total hip replacement; tri-n-butylborane.

Figure 1

Initial osteoblast attachment to culture wells after 24 h of culture with three different bone cement extracts. (A) The number of cells attached to the culture wells after one day of culture was evaluated by manually counting cells using a hemocytometer. Data shown are mean ± SD (n = 3). Significant differences between the three groups are shown (one-way ANOVA followed by Bonferroni correction, * p < 0.05. (B) Representative fluorescence microscopy images of initially attaching osteoblasts after 24 h of culture stained with rhodamine phalloidin for actin filaments (red) and 4′,6-diamidino-2-phenylindole (DAPI) for nuclei (blue) at low magnification.

Figure 2

Evaluation of the proliferation of osteoblasts exposed to three different bone cement extracts. (A) The density of propagated cells after 3 and 5 days of culture was evaluated by manually counting cells using a hemocytometer. (B) Representative fluorescence microscopy images of propagated osteoblasts after 3 days of culture stained with rhodamine phalloidin for actin filaments (red) and 4′,6-diamidino-2-phenylindole (DAPI) for nuclei (blue) at low magnification. (C) BrdU incorporation per cell measured at days 3 and 5. Data shown are mean ± SD (n = 3). Significant differences between the three groups are shown (one-way ANOVA followed by Bonferroni correction, ** p < 0.01).

Figure 3

Spreading and cytoskeletal arrangement of osteoblasts exposed to three different bone cement extracts 1 and 3 days after seeding. Representative high magnification confocal microscopy images of the spreading behavior of osteoblasts stained with rhodamine phalloidin for actin filaments (red), DAPI for nuclei (blue), and vinculin (green) after (A) 1 day and (B) 3 days of culture (top panels). Cell morphometry analysis of the images (histograms shown below). Data shown are mean ± SD (n = 5). Significant differences between the three groups are shown (one-way ANOVA followed by Bonferroni correction, * p < 0.05, ** p < 0.01).

Figure 4

Differentiation of osteoblasts exposed to three different bone cement extracts. Alkaline phosphatase (ALP) evaluation of osteoblasts exposed to three different bone cement extracts 3 and 7 days after seeding. (A) Colorimetric quantification of ALP activity. (B) Representative images of ALP staining. Data shown are mean ± SD (n = 3). Significant differences between the three groups are shown (one-way ANOVA followed by Bonferroni correction, ** p < 0.01).

Figure 5

Evaluation of TRAP activity of osteoclasts after 3 days of bone cement extract exposure. (A) Representative images of TRAP staining. (B) The number of TRAP-positive multinucleated cells as determined by the number of cells containing 3 or more nuclei. Data shown are mean ± SD (n = 3). Significant differences between the three groups are shown (one-way ANOVA followed by Bonferroni correction, ** p < 0.01).

Figure 6

Evaluation of osteoclast activity stimulated by three different bone cement extracts. Relative gene expression levels of osteoclast TRAP, NFATc1, and cathepsin K after 1 and 3 days of exposure to bone cement extracts. Data shown are mean ± SD (n = 3).