N-acetyl cysteine (NAC)-mediated detoxification and functionalization of poly(methyl methacrylate) bone cement☆
Introduction
Bone fracture and degenerative changes in joints are quite common among patients with osteoporosis and rheumatoid arthritis. Approximately 30,0000 incidents of hip fracture alone occur each year in the US, and the annual cost of treatment is estimated to be about $13.8 billion [1]. Metallic implants used to repair or reconstruct bones and joints are fixed in bones using poly(methyl methacrylate) (PMMA) resin-based bone cement materials. However, loosening of implants remains the most important complication, resulting in a high incidence of revision surgeries [2], [3], [4], [5], [6], [7]. Due to the recent surge of treatment cost and universally advancing ageing society, greater emphasis is being placed on identifying the etiology and preventing this undesirable outcome [8].
Bone cement fills the gap between the implant and the existing bone to produce mechanical interlocking. It does not adhere to the existing bone or induce new bone formation due to its poor osteoconductivity; instead, fibrous soft tissue forms around the bone cement [9], [10], [11], [12]. Moreover, bone cement can induce multiple adverse reactions, including impaired bone remodeling capability, necrosis, fibrosis, and histiocytosis [13], [14]. At the cellular level, bone cement inhibits the proliferation and function of osteoblastic cells [15], [16], [17], and induces cellular apoptosis and necrosis [18]. It has also been known to have severe adverse effects on systemic functions, collectively termed as “bone cement implantation syndrome” or BCIS. This syndrome is characterized by hypotension, hypoxemia, cardiac arrhythmias, cardiac arrest, or any combination of these, and is even known to cause an immediate death in some cases (0.6–1% in some recipient groups) [19], [20]. The mechanism underlying such cardiovascular malfunctions remains unknown.
Free radicals generated from acrylic resin polymerization induce oxidative stress and directly cause cytotoxicity [21], [22], [23]. In addition, cells exposed to acrylic ingredients produce reactive oxygen species (ROS), including free radicals [8], [18], [24]. Glutathione is a cysteine derivative primarily located in the cell membrane, and the glutathione-mediated redox cycle is the most important removal system for such exogenous and endogenous free radicals [25], [26]. N-acetyl cysteine (NAC), the N-acetyl derivative of cysteine, is also known as an antioxidant. NAC is easily deacetylated into cysteine, which is an important precursor of glutathione [27] and helps promote the cellular glutathione system [28], [29]. NAC also directly acts as a strong oxidant scavenger by neutralizing free radicals with a hydrogen atom of its thiol moiety [29].
We hypothesized that incorporation of NAC in the liquid monomer of a PMMA bone cement significantly reduces the cement's cytotoxicity, via the in situ interaction of the NAC and the free radicals generated when the cement polymerizes, and results in significant restoration of the osteoblastic and osteogenic activities of the cement. Also, we investigated the viability, gene expression and mineralizing capability of rat bone marrow-derived osteoblastic cells cultured on NAC-incorporated bone cement; the osteoconductivity of the cement, under in vivo conditions; and the effects of NAC on the mechanical and setting properties of the cement. Furthermore, we sought to gain understanding of the mechanisms to which the NAC-mediated osteogenic functions of the cement may be attributed.
Section snippets
Bone cement preparation
Untreated control PMMA bone cement was prepared by mixing the recommended ratio of powder and liquid (0.34 g powder and 173.15 μl liquid, Endurance MV, DePuy Orthopaedics, Warsaw, IN) for 25 s. N-acetyl cysteine (NAC)-supplemented bone cement was prepared by mixing 0.34 g powder, 173.15 μl liquid containing N-acetyl cysteine (NAC) giving final concentrations of 10, 25, or 50 mm. A stock solution of 1 mol NAC was prepared in HEPES buffer and the pH was adjusted to 7.2.
Bone cement extract preparation
The bone cement was mixed in the
Complete suppression of osteoblastic phenotypes by the bone cement and its prevention by NAC
Osteoblastic cells cultured on untreated control bone cement for 14 days showed an almost complete suppression of alkaline phosphatase (ALP) activity (Fig. 1A). In contrast, 90% of the area of the cultures on the 25 mm NAC-supplemented bone cement was found to be ALP-positive (p < 0.001), which was equivalent to the value recorded for cultures on polystyrene (p > 0.05). Addition of 25 mm NAC to the culture medium also restored the ALP activity, although the effect of NAC was less as compared to when
Discussion
The suppression of osteoblastic function on the untreated bone cement was more pronounced than we had expected and was supported by the sustained level of free radicals generated in the material at least for 20 days. Although cell viability 24 h after seeding onto the bone cement was 45.7%, viability after a longer culture period may be poorer. Our data suggested that the NAC supplementation prevents the bone cement-induced osteoblastic cell death and functionalizes the material, i.e., an
Conclusions
In this study, it was determined that incorporation of the amino acid derivative N-acetyl cysteine (NAC) in the liquid monomer of a PMMA bone cement reduced the cytotoxicity of the cement and rendered it osteoconductive. Completely suppressed osteoblastic phenotypes on the untreated bone cement were restored to the normal level by NAC incorporation. Bone volume formed around this NAC-incorporated bone cement was threefold greater than that around the control bone cement. The in vivo strength of
Acknowledgement
This work was supported by JAMSEA and Japan Medical Materials (JMM) Corporation. This study was conducted in a facility constructed with support from the Research Facilities Improvement Program, grant No. C06RR014529, of the National Center for Research Resources, National Institute of Health.
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This work was partially supported by JAMSEA and Japan Medical Materials (JMM) Corporation. This study was conducted in a facility constructed with support from the Research Facilities Improvement Program, Grant No. C06RR014529, of the National Center for Research Resources, National Institute of Health.