In vitro studies on the effect of particle size on macrophage responses to nanodiamond wear debris

Abstract

Nanostructured diamond coatings improve the smoothness and wear characteristics of the metallic component of total hip replacements and increase the longevity of these implants, but the effect of nanodiamond wear debris on macrophages needs to be determined to estimate the long-term inflammatory effects of wear debris. The objective was to investigate the effect of the size of synthetic nanodiamond particles on macrophage proliferation (BrdU incorporation), apoptosis (Annexin-V flow cytometry), metabolic activity (WST-1 assay) and inflammatory cytokine production (qPCR). RAW 264.7 macrophages were exposed to varying sizes (6, 60, 100, 250 and 500 nm) and concentrations (0, 10, 50, 100 and 200 μg ml⁻¹) of synthetic nanodiamonds. We observed that cell proliferation but not metabolic activity was decreased with nanoparticle sizes of 6–100 nm at lower concentrations (50 μg ml⁻¹), and both cell proliferation and metabolic activity were significantly reduced with nanodiamond concentrations of 200 μg ml⁻¹. Flow cytometry indicated a significant reduction in cell viability due to necrosis irrespective of particle size. Nanodiamond exposure significantly reduced gene expression of tumor necrosis factor-α, interleukin-1β, chemokine Ccl2 and platelet-derived growth factor compared to serum-only controls or titanium oxide (anatase 8 nm) nanoparticles, with variable effects on chemokine Cxcl2 and vascular endothelial growth factor. In general, our study demonstrates a size and concentration dependence of macrophage responses in vitro to nanodiamond particles as possible wear debris from diamond-coated orthopedic joint implants.

Introduction

Data from the American Academy of Orthopedic Surgeons show that more than 418,000 total and partial knee replacements and 328,000 total and partial hip replacements are performed in the US each year, and the number of total knee replacements and total hip replacements performed in the US is expected to leap by 673% and 174%, respectively, by the year 2030 [1]. Wear of articulating surfaces involving cobalt–chromium–molybdenum (CoCrMo) alloy against polyethylene (components in the majority of hip and knee implants) has been cited as a dominant factor limiting the long-term success of the implants [2], [3]. Wearing and the resultant generation of wear debris particles can lead to mechanical instability, decreased joint mobility, increased pain, deleterious biological responses, osteolysis, and ultimately component loosening and implant failure [2], [3], [4]. Large debris pieces are normally sequestered by fibrous tissue, while small debris is phagocytosed by macrophages and monocytes, which may release cytokines that result in inflammation. The inflammation cascade also leads to the recruitment of activated osteoclasts at the bone–implant interface and to bone resorption around the implant. One solution for this problem of osteolysis caused by wear debris is to coat harder materials, such as diamond, on the articulating surfaces to improve wear resistance and to reduce the number and size of debris particles generated.

Studies have shown that less wear occurs with metal-on-metal hip prostheses, up to 100 times less than that of polyethylene-on-metal, and CoCrMo metal articulation results in smaller debris particles than metal-on-polyethylene articulation [5], [6], [7]. The CoCrMo debris from a metal-on-metal articulation were shown to be in the range of 6–744 nm, with an average size of 42 nm [6], whereas polyethylene particles ranged from 0.1 to 5 μm (100–5000 nm) [8]. As the metal wear-particles are relatively smaller than polyethylene particles, they may be less prone to inflammation and osteolytic reaction. A study investigating bone and tissue reactions to metal debris in several metal-on-metal components revealed that there were fewer macrophages and giant cells than typically seen in tissues around metal-on-polyethylene joints [2]. Unfortunately, there are concerns and debate associated with metal sensitivity and metallosis, the release of alloy constituents into the surrounding tissue. The term “metallosis” has also been used to describe the intraoperative findings of gross metallic debris and blackening of periprosthetic tissue [9]. Metal sensitivity, which affects approximately 10–15% of the general population, is much higher among patients with failed implants [10], [11], [12].

Nanostructured diamond (NSD) coatings or diamond-like carbon coatings have been shown to greatly enhance the wear resistance and to prevent leaching of metallic ions from orthopedic and dental implants into the body [13], [14]. For example, NSD coatings improve the smoothness and wear characteristics of the femoral head component of total hip replacements, and hence minimize wear debris formation and increase implant longevity [14], [15], [16]. NSD coatings developed in our laboratory have shown promise as potential wear-resistant coatings on Ti–6Al–4V for use in temporomandibular joint prostheses [16], [17], [18] and on CoCrMo for hip joint prostheses [19]. However; the brittleness/delamination of a thin diamond coating may lead to the formation of wear-debris of nanodiamonds from diamond-on-diamond and diamond-on-polyethylene couples.

The average particle size of diamond-on-diamond debris is expected to be considerably smaller than polyethylene debris, due to the increased material hardness. In addition, hip simulator studies [20] involving diamond–diamond couples have shown very marked reduction in wear debris volume (<10⁻⁴ mm³ year⁻¹), compared to first-generation CoCrMo–CoCrMo couples (1–5 mm³ year⁻¹) and to polyethylene couples (50–100 mm³ year⁻¹). The reduced wear volume and particle size expected for diamond articulation is a major advantage over conventional orthopedic bearings. However, a very low wear volume itself is not the only key factor governing the long-term clinical outcome of a total joint replacement. The number, size, or morphology and biological response to wear particles released are also important factors. In addition, there exists considerable discussion over the possible distribution of these smaller nanoparticles in the body, and their biological effects on cells and tissues. The objective of this study was to evaluate systematically the response of inflammatory cells, macrophages, to synthetic nanodiamond particles of various sizes as potential wear debris from diamond-on-diamond couples. Recently, synthetic nanodiamond particles have attracted much attention in therapeutic delivery and bio-imaging applications due to their innate inertness and emission of fluorescence from nitrogen vacancies (N-V color centers) [21], [22], [23], [24], [25]. A few studies have reported the cytocompatibility of nanodiamond particles (2–10 nm), produced by an explosive detonation technique, from the standpoint of cell viability using the MTT assay, ATP production and ROS measurement with different cell types in vitro [26], [27], [23]. An in vivo study to investigate the fate of nanodiamonds (50 nm diameter) in mice has shown accumulation of 60% of injected particles in the liver after 0.5 h of post-dosing and the rest in the spleen and lung [28]. However, the effect of the size and concentration of nanodiamond particles when exposed to phagocytes such as macrophages and their potential activation from a standpoint of bone resorption/implant failure has not yet been explored in detail.

An ongoing concern in conventional total joint replacement prostheses is osteolysis and aseptic loosening induced by wear-debris originating from the interfaces of articulating components [29], [30], [31], [32]. Macrophages and multinuclear giant cells (MNGCs, formed by the fusion of macrophages) are the primary scavengers of wear particles and their response appears to depend on the nature, size and concentration of the particles accumulated [3], [4]. Polyethylene debris surrounded by macrophages and phagocytosis of debris by macrophages was frequently observed at the interface tissues [33]. Phagocytic cells engulf wear debris and become activated, releasing pro-inflammatory cytokines, degradative enzymes and other factors that stimulate osteoclasts [33], [34], [35]. For a diamond-on-polyethylene couple, polyethylene has much lower hardness compared to diamond and would account for essentially all of the wear-particles generated. However, for a diamond-on-diamond couple, diamond wear-particles are expected to be produced, and the effects of these particles on inflammatory cells must be evaluated. A limitation is that the amount of nanodiamond debris generated from a wear couple in a simulator is expected to be negligible due to its superior hardness, smoothness and wear-resistant properties [16], [36]. Moreover, the particles produced in simulators or by other means do not consistently resemble wear debris particles from joint implants in vivo [37]. Therefore, synthetic nanodiamond particles of different sizes (6–500 nm) and varying concentrations (10–200 μg ml⁻¹) were utilized to interact with macrophage cells in the present study. Macrophage response to titanium dioxide (anatase) particles (∼8 nm average size), a well-known nanoparticle, was evaluated for the purpose of comparison.